- Email: [email protected]
Synthesis, crystal structure and photo-induced DNA cleavage activity of ternary copper(II)-thiosemicarbazone complexes having heterocyclic bases
Inorganica Chimica Acta 357 (2004) 2315–2323 www.elsevier.com/locate/ica
Synthesis, crystal structure and photo-induced DNA cleavage activity of ternary copper(II)-thiosemicarbazone complexes having heterocyclic bases q Anitha M. Thomas, Anil D. Naik, Munirathinam Nethaji, Akhil R. Chakravarty
*
Department of Inorganic and Physical Chemistry, Indian Institute of Science, Sir C.V. Raman Avenue, Bangalore 560012, India Received 1 October 2003; accepted 31 January 2004 Available online 21 February 2004
Abstract New ternary copper(II) complexes of formulations [Cu(Ph-tsc)B] (B ¼ 1,10-phenanthroline, phen (1); dipyridoquinoxaline, dpq (2); dipyridophenazine, dppz (3); Ph-H2 tsc, salicylaldehyde-N(4)-phenylthiosemicarbazone) and [Cu(Me-tsc)(phen)] (4, Me-H2 tsc, salicylaldehyde-N(4)-methylthiosemicarbazone) are prepared, and their DNA binding and cleavage properties studied. Complex 1 has been characterized by single crystal X-ray crystallography. The molecular structure shows a distorted square pyramidal (4 + 1) geometry of the complex with the dianionic NSO-donor N(4)-phenyl-substituted thiosemicarbazone binding at the basal plane and the NN-donor planar heterocyclic base (phen) displaying axial–equatorial coordination. The one-electron paramagnetic complexes exhibit axial EPR spectra and show a d–d band near 580 nm for the phen and near 720 nm for the dpq, dppz complexes in their electronic spectra in DMF. The complexes show quasireversible cyclic voltammetric response near 0.08 V vs. SCE in DMF–0.1 M TBAP assignable to the Cu(II)/Cu(I) couple. The Ph-tsc complexes display good binding propensity to calf thymus (CT) DNA. They also show oxidative cleavage of supercoiled (SC) pUC19 DNA in dark under aerobic condition in the presence of mercaptopropionic acid. The complexes exhibit light-induced DNA cleavage activity at 312 and 532 nm. Mechanistic investigations reveal DNA minor groove binding for the phen and dpq complexes, and major groove binding for the dppz species. The complexes are cleavage inactive under argon atmosphere. In the ternary structure, the thiosemicarbazones, dpq and dppz act as photosensitizers, while the planar heterocyclic bases are binder to DNA. The mechanistic pathways involved and the role of metal in the DNA cleavage reactions are discussed. Ó 2004 Elsevier B.V. All rights reserved. Keywords: Ternary copper(II) complex; Thiosemicarbazones; Heterocyclic bases; DNA Photocleavage; Crystal structure
1. Introduction Designing transition metal complexes, that can bind and cleave DNA under physiological conditions, is of importance in the development of diagnostic agents for medicinal applications [1–10]. The DNA cleavage reactions generally proceed via three major pathways, viz. oxidative strand cleavage by abstraction of sugar hydrogen atom(s), hydrolytic cleavage involving the phosphate group, and by base oxidations primarily directed at the guanine base. Among different methodolq Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ica.2004.01.031. * Corresponding author. Tel.: +918022932533; fax: +918023600683. E-mail address: [email protected] (A.R. Chakravarty).
0020-1693/$ - see front matter Ó 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ica.2004.01.031
ogies adopted for inducing DNA cleavage by synthetic nucleases, the one based on photo-irradiation has received current attention with the advent of photodynamic therapy (PDT) of cancer [11–15]. The PDT process generally requires a photosensitizer (drug), a visible light source (preferably red light), and oxygen. The drug on photo-excitation is promoted to its excited singlet state. It then populates triplet states due to rapid intersystem crossing followed by activation of triplet oxygen to its singlet state. Porphyrin derivatives have largely been used as photosensitizers for medicinal applications in PDT [14,15]. The present work stems from our continued interest to design copper-based transition metal complexes cleaving DNA on photo-irradiation. Although there are reports [16–20] of copper complexes cleaving DNA hydrolytically or in an oxidative manner
2316
A.M. Thomas et al. / Inorganica Chimica Acta 357 (2004) 2315–2323
in the presence of a reducing agent, the chemistry of copper complexes showing visible light-induced photocleavage is virtually unexplored [21,22]. We have reported that non-porphyrinic copper(II) complexes can cause significant cleavage of supercoiled DNA in the presence of suitable photosensitizer(s) in a ternary structure [22]. Our observation of copper(II)-assisted visible light-induced DNA photo-cleavage in a ternary complex is important as the copper(II)–porphyrin complexes, in contrast, are known to be inefficient DNA photo-cleaver due to significant effect of the metal center in quenching the porphyrin 3 (p–p ) state life time [23]. The utility of thiosemicarbazone-copper(II) complexes in DNA binding and in vitro studies has recently been reported by Baldini and coworkers [24]. We have probed the DNA binding and photocleavage properties of a series of ternary copper(II) complexes [Cu(Ph-tsc)B] (B ¼ phen (1); dpq, (2); dppz (3)) and [Cu(Metsc)(phen)] (4) (Chart 1), containing N(4)-mono-substituted thiosemicarbazones and planar phenanthroline bases. Our choice for the sulfur-containing thiosemicarbazones is based on the fact that thio or thione moieties are known to show efficient intersystem crossing to the triplet state on photo-irradiation [22,25–27]. Such an excited state with a longer lifetime can either cause damage to DNA directly or can activate oxygen from its stable triplet (3 O2 ) to the cytotoxic singlet state (1 O2 ). The choice of thiosemicarbazones is based on their significant pharmacological properties. Besides, several thiosemicarbazone transition metal complexes are known to exhibit potential antitumor activities [28–30]. It has been proposed that the cellular oxidative chemistry, involving copper thiosemicarbazones in the absence of photo-irradiation, is similar to those of iron(II) bleomycin or copper(I) phenanthroline complexes [31– 33]. In our scheme of work, we have used thiosemicarbazones (Ph-tsc, Me-tsc) as potential photosensitizers in ternary copper(II) complexes in which the role of heterocyclic bases, with extended planar aromatic ring(s),
Chart 1.
viz. 1,10-phenanthroline (phen), dipyridoquinoxaline (dpq) or dipyridophenazine (dppz), is to primarily bind to double-stranded DNA. In addition, the dpq and dppz ligands can also act as photosensitizer with their respective quinoxaline and phenazine moieties. Significant results of the present study include the observation of efficient DNA cleavage by the dpq and dppz complexes on photo-irradiation at green light (k, 532 nm) and different DNA-groove selectivity of the complexes.
2. Experimental 2.1. Chemical reagents All reagents and chemicals of analytical grade were purchased from commercial sources. Solvents used for spectral and electrochemical measurements were purified by standard procedures. Salicylaldehyde, N(4)-phenylthiosemicarbazide, and N(4)-methylthiosemicarbazide were obtained from Aldrich (USA). Copper(II) acetate hydrate and 1,10-phenanthroline (phen) were purchased from SD Fine Chemicals, Mumbai. Calf thymus (CT) DNA and supercoiled (SC) pUC19 DNA (cesium chloride purified) were from Bangalore Genie (India). Agarose (molecular biology grade), ethidium bromide (EB) and distamycin were obtained from Sigma (USA). Tris–HCl buffer solution was prepared using deionized, sonicated triple distilled water. Complexes [Cu(dpq) (NO3 )2 ], [Cu(dppz)(NO3 )2 ] and [Cu(phen)2 (H2 O)] (ClO4 )2 , used for control experiments, were prepared by reported methods [34,35]. The thiosemicarbazones (PhH2 tsc and Me-H2 tsc) were prepared by a literature method [36]. The heterocyclic bases, dipyrido[3,2-d:20 ,30 f]quinoxaline (dpq) and dipyrido[3,2-a:20 ,30 -c]phenazine (dppz), were prepared using 1,10-phenanthroline as a precursor by literature procedures [37,38]. 2.2. Measurements The elemental analytical data were obtained from a Heraeus CHN–O rapid instrument. The infrared, electronic, and EPR spectral data were recorded using Bruker Equinox 55, Hitachi U-3400, and Varian E-109 X-band spectrometers, respectively. Magnetic susceptibility data at room temperature were obtained from a George Associates Inc. (Berkeley, CA) Lewis-coil force magnetometer. Hg[Co(NCS)4 ] was used as a standard. Cyclic voltammetric measurements were done at 25 °C on a EG&G PAR Model 253 Versa Stat potentiostat/ galvanostat with electroanalytical software 270 using a three electrode setup consisting of a glassy carbon working, platinum wire auxiliary, and a saturated calomel reference (SCE) electrode. Tetrabutylammonium perchlorate (TBAP, 0.1 M) was used as supporting electrolyte. The Fe(III)/Fe(II) couple of ferrocene as a
A.M. Thomas et al. / Inorganica Chimica Acta 357 (2004) 2315–2323
standard was observed at 0.4 V vs. SCE in DMF–0.1 M TBAP. 2.3. Synthesis of the compounds 2.3.1. [Cu(Ph-tsc)(phen)] (1) and [Cu(Me-tsc) (phen)] (4) The complexes were prepared by a general method, in which copper(II) acetate hydrate (0.2 g, 0.5 mmol) was reacted with 1,10-phenanthroline (phen, 0.2 g, 1.0 mmol) and the thiosemicarbazone (Ph-H2 tsc, 0.27 g; Me-H2 tsc, 0.21 g; 1.0 mmol) in 25 mL MeOH. The solution was refluxed for 3 h, followed by cooling to an ambient temperature. The solid thus obtained was washed with cold methanol and dried under vacuum over P4 O10 (Yield: 65%). Anal. Calc. for C26 H19 N5 OSCu (1): C, 60.86; H, 3.73; N, 13.64. Found: C, 60.72; H, 3.94; N, 13.73%. IR (KBr phase, cm1 ): 3398w, 3056w, 1600s, 1534s, 1495s, 1421s, 1314m, 840m, 736m (s, strong; m, medium; w, weak). Anal. Calc. for C21 H17 N5 OSCu (4): C, 55.93; H, 3.80; N, 15.53. Found: C, 55.76, H, 3.57; N, 15.63%. IR (KBr disc, cm1 ): 3231w, 3050w, 1600s, 1513s, 1448m, 1403m, 1340m, 1198m, 849m, 724m. 2.3.2. [Cu(Ph-tsc)(dpq)] (2) and [Cu(Ph-tsc)(dppz)] (3) The complexes were synthesized using a general procedure in which a hot methanol solution (60 °C, 25 mL) of the thiosemicarbazone ligand (Ph-H2 tsc, 0.27 g, 1.0 mmol) was initially reacted with copper(II) acetate hydrate (0.2 g, 0.5 mmol) taken in methanol (5 mL) under magnetic stirring for 30 min. The solution was cooled to an ambient temperature and the heterocyclic base (1.0 mmol; dpq, 0.23 g; dppz, 0.28 g) dissolved in CHCl3 (20 mL) was added to the solution that was stirred for a further period of 2 h. Diethyl ether was added to the solution to precipitate the solid which was isolated, washed with CHCl3 , and finally dried in vacuo over P4 O10 . Yield: 60%. Anal. Calc. for C28 H19 N7 OSCu (2): C, 59.51; H, 3.38; N, 17.35. Found: C, 59.67; H, 3.28; N, 17.21%. IR (KBr phase, cm1 ): 3425w, 3065w, 1600m, 1573m, 1483m, 1385s, 1078m, 810m, 736m. Anal. Calc. for C32 H21 N7 OSCu (3): C, 62.47; H, 3.44; N, 15.93. Found: 62.54; H, 3.57; N, 16.15%. IR (KBr disc, cm1 ): 3446w, 3076w, 1600s, 1492m, 1373m, 1098m, 813m, 754m, 730m. 2.4. DNA binding and cleavage experiments The concentration of calf thymus (CT) DNA was determined from the absorption intensity at 260 nm with a molar extinction coefficient value of 6600 M1 cm1 in Tris–HCl/NaCl buffer (pH 7.2) [39]. In a typical binding experiment, a 2.0 mL solution of ethidium bromide (EB) was added to a volume of 100 lL of 125 lM CT DNA solution to get the maximum fluorescence intensity of
2317
EB (excitation at 510 nm; emission at 601 nm). Aliquots of 10 mM solution of the complex in DMF were added to the EB bound CT DNA solution and the fluorescence intensity had been measured after each addition until a 50% reduction of the intensity was observed. The fluorescence intensities were plotted against the complex concentration to get a slope that gave the relative measure of the extent of binding of the complex to the CT DNA. A control experiment was also done with the EB in the absence of CT DNA and the observed intensities were plotted against the complex concentration. DNA cleavage activity of the complexes was monitored by agarose gel electrophoresis in the presence or absence of a reducing agent under dark or illuminated conditions. In a typical ‘‘chemical nuclease’’ activity study, supercoiled (SC) pUC19 DNA (6 lL, 500 ng) in Tris–HCl buffer (50 mM, pH 7.2) containing 50 mM NaCl (2 lL) was treated with the ternary copper(II) complex (2 lL, 30 lM) and mercaptopropionic acid (2 lL, 50 mM) followed by dilution with the buffer to a total volume of 20 lL in a dark chamber. The samples were then incubated for 1 h at 37 °C, added loading buffer containing 25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol (3 lL) and loaded on 0.8% agarose gel containing 1.0 lg/mL ethidium bromide. Electrophoresis was carried out at 40 V for 2 h in Tris-acetateEDTA (TAE) buffer. Bands were visualized by UV light and photographed followed by the estimation of the intensity of the DNA bands using UVITEC Gel Documentation system. Due correction was made for the low level of NC (nicked circular) DNA present in the original sample and the low affinity of ethidium bromide binding to SC DNA in comparison to its NC form [37]. Inhibition reactions were carried out under dark condition by prior incubation of the SC DNA with DMSO (5 lL) or distamycin (2 lL, 100 lM) for 10 min before the addition of the ternary complex and the reducing agent (MPA), and then subjected for further incubation and gel electrophoresis using the above procedures. The photo-induced pUC19 DNA cleavage studies were done using monochromatic UV (k ¼ 312 nm, 96 W (total wattage) transilluminator UVITEC make) and visible (k ¼ 532 nm, mercury lamp of 125 W of commercial source) light in the absence of MPA. For mechanistic investigations, the inhibition reactions were carried out at 312 nm using different reagents like sodium azide (10 mM), DMSO (4 lL), mannitol (100 mM) and sodium formate (100 mM). For the D2 O experiment, this solvent was used for dilution to 18 lL. The photo-cleavage experiment was also done under an argon atmosphere. 2.5. X-ray crystallography Greenish-brown crystals of 1 H2 O were grown by a diffusion technique in which diethyl ether was layered above the complex solution in MeOH–CHCl3 mixture
2318
A.M. Thomas et al. / Inorganica Chimica Acta 357 (2004) 2315–2323
Table 1 Selected crystallographic data for [Cu(Ph-tsc)(phen)] (1 H2 O) 1 H2 O Empirical formula Crystal size Crystal color Crystal morphology Crystal system Space group Unit cell dimensions a (A) b (A) c (A)
C26 H21 CuN5 O2 S 0.2 0.14 0.1 greenish-brown rectangular monoclinic Cc 17.603(4) 11.435(2) 12.344(3) 107.089(3) 2375.1(8) 4 1.485 1.042 1092 0.71073 293(2) 4203 3935 316 0.0442 0.1106 0.0467 0.1125 1.021 0.000 0.414 and )0.264
b (°) 3 ) V (A Z Dcalc (g cm3 ) l(Mo) (mm1 ) F ð0 0 0Þ k (Mo Ka) (A) T (K) Reflections collected Reflections observed [I > 2rðIÞ] Parameters Ra (observed data) Rw b (observed data) R (all data) Rw (all data) Goodness-of-fit on F 2 Shift/e.s.d. (maximum) 3 ) Largest difference peak and hole (e A P P a R¼ P kFo j jFc k= jFP o j. b wR ¼ f ½wðFo2 Fc2 Þ2 = ½wðFo Þ2 g1=2 ;w ¼ ½r2 ðFo Þ2 þ ðAP Þ2 þ BP 1 , where P ¼ ðFo2 þ 2Fc2 Þ=3; A and B values are 0.0779 and 0.0000.
(5:1 v/v). The source of water could be methanol used for crystallization of the complex. The unit cell parameters and the intensity data were collected on a Bruker SMART APEX CCD diffractometer, equipped with a fine focus 1.75 kW sealed tube Mo Ka X-ray source, with increasing x (width of 0.3°/frame) at a scan speed of 15 s/frame. The SMART software was used for data
acquisition and the SAINT software for data abstraction. Absorptions corrections on the data were made using SADABS [40]. Structure was solved and refined with S H E L X programs [41]. Hydrogen atoms in their calculated positions were refined using a riding model. The non-hydrogen atoms were refined anisotropically. The crystal structure showed the presence of one lattice water molecule in the asymmetric unit. Selected crystallographic data are given in Table 1. Perspective view of the complex was obtained by ORTEP [42].
3. Results and discussion 3.1. Synthetic and general properties The ternary copper(II) complexes 1–4 have been prepared in high yield from a reaction of copper(II) acetate hydrate with the NSO-donor thiosemicarbazone ligand and the NN-donor heterocyclic base. Heterocyclic bases chosen are 1,10-phenanthroline (phen) and its derivatives dipyridoquinoxaline (dpq) and dipyridophenazine (dppz). The presence of extended aromatic ring(s) is expected to enhance the binding propensity with DNA. We have also chosen two thiosemicarbazone ligands having mono-N(4)-substituted methyl or phenyl group to explore their effect on the cleavage activity. The tridentate thiosemicarbazone and didentate heterocyclic base combination gives stable five coordinated structure for the copper(II) center in 1–4. Selected physicochemical data of the complexes are given in Table 2. The one-electron paramagnetic complexes show X-band axial EPR spectra in DMF–toluene glass at 77 K giving gk > g? , indicating a fdx2 y 2 g1 ground state in a square pyramidal geometry. The Ak value for the phen complexes is significantly higher than that for the dpq and dppz complexes. This is indicative of significant distortion from the square pyramidal geometry in 2 and 3 [43,44]. The phen complexes display a
Table 2 Physicochemical data on complexes [Cu(Ln )B] (1–4) kmax / nm ( / M1 cm1 )a
EPRb
d–d band
CT band
gk (104 Ak /cm1 )
g?
1
577 (155)
2.17 (175)
2
720 (105)
3
726 (110)
4
578 (150)
389 (12 400), 318 (17 400), 289 (20 000) 340 (10 250), 397 (3100), 326 (12 100) 386 (3550), 338 (11 450), 322 (14 400) 378 (10 400), 323 (14 600), 309 (15 800)
Complex
a
leff =ðlB Þc
Cu(II)/Cu(I) coupled E1=2 =VðDEp =mVÞ
ipc =ipa
1.98
1.84
0.079 (145)
1.1
2.26 (150)
2.06
1.87
0.080 (140)
1.2
2.26 (150)
2.06
1.82
0.082 (135)
0.9
2.17 (175)
1.98
1.81
0.077 (186)
1.1
In DMF–Tris buffer (pH 7.2, 1:1 v/v). In DMF–toluene glass at 77 K. c At 298 K. d In DMF–0.1 M TBAP. E1=2 ¼ 0:5ðEpa þ Epc Þ, DEp ¼ Epa Epc , where Epa and Epc are anodic and cathodic peak potentials, respectively. Scan rate: 50 mV s1 . ipc and ipa are cathodic and anodic peak currents, respectively. b
A.M. Thomas et al. / Inorganica Chimica Acta 357 (2004) 2315–2323
d–d band in the electronic spectra near 580 nm. A significant shift of this band to lower energy is observed for the dpq and dppz complexes, possibly due to the structural differences among the complexes arising from the extended aromatic rings of these ligands [44]. While the phen complexes show one low energy ligand to copper(II) charge transfer (LMCT) band near 400 nm, the dpq and dppz complexes display two such bands. The lower energy band could be assignable to the sulfur to copper(II) CT transition, while the other may involve the heterocyclic base. The complexes are redox active and show quasireversible cyclic voltammetric response due to the Cu(II)/Cu(I) couple near 0.08 V vs. SCE in DMF–0.1 M TBAP with DEp values ranging between 135 and 186 mV at a scan rate of 50 mV s1 with an ipc =ipa ratio of unity, where ipc and ipa are cathodic and anodic peak currents, respectively. 3.2. Crystal structure Complex [Cu(Ph-tsc)(phen)] H2 O (1 H2 O) has been structurally characterized by single crystal X-ray crystallography. An ORTEP view of the complex is shown in Fig. 1. Selected bond distances and angles are given in Table 3. In the ternary structure, the copper atom is bonded to the thiosemicarbazone ligand showing NSObinding mode at the basal plane in a square pyramidal (4 + 1) geometry in which the NN-donor phen displays axial–equatorial coordination. The copper atom is dis above the N2 SO basal plane. The placed 0.24(1) A trigonality parameter (s) value of 0.32 is indicative of distortion from the square pyramidal geometry [45]. The thiosemicarbazone ligand binds the copper(II) center in a dianionic tridentate fashion with the phenolate oxygen O(1), imine nitrogen N(3) and the sulfur S(1) forming a –N@C(S )-NHPh moiety. The imine bond formation is evidenced from the N(3)–C(19) and N(4)–C(20) dis The dihedral angle tances of 1.283(7) and 1.292(7) A.
Fig. 1. ORTEP view of the complex in 1.H2 O with atom numbering scheme and 50% probability thermal ellipsoids.
2319
Table 3 and angles (°) for [Cu(Ph-tsc)(phen)] Selected bond lengths (A) (1 H2 O) with estimated standard deviations in their parentheses Bond lengths Cu(1)–S(1) Cu(1)–O(1) Cu(1)–N(1) Cu(1)–N(2) Cu(1)–N(3)
Bond angles 2.2489(13) 1.949(3) 2.256(4) 2.042(3) 1.962(3)
S(1)–Cu(1)–O(1) S(1)–Cu(1)–N(1) S(1)–Cu(1)–N(2) S(1)–Cu(1)–N(3) O(1)–Cu(1)–N(1) O(1)–Cu(1)–N(2) O(1)–Cu(1)–N(3) N(1)–Cu(1)–N(2) N(1)–Cu(1)–N(3) N(2)–Cu(1)–N(3)
158.35(11) 103.64(10) 92.46(10) 84.91(10) 97.99(14) 91.24(12) 92.00(13) 77.69(14) 100.79(13) 176.59(15)
between the mean planes of the five and six member chelate rings of the thiosemicarbazone ligand is 15.62°. Near planar arrangement of the chelate rings and the presence of conjugation in the Schiff base could have significant effect on its photosensitizing ability. 3.3. DNA binding and chemical cleavage activity Fluorescence spectral technique has been used to determine the relative binding propensity of the ternary complexes to CT DNA by measuring the emission intensity of ethidium bromide (EB) bound to DNA [46]. Bis(phen)copper(II) complex has been used as a standard. EB does not show any emission in the Tris-buffer medium due to fluorescence quenching by the solvent molecules [47]. However, in the presence of CT DNA, it shows enhanced emission intensity due to its intercalative binding to DNA. Addition of copper complexes results in the competitive binding of the complexes to DNA, thus reducing the emission intensity with either a displacement of the EB from the bound to the free state or the bound copper(II) complex quenching the emission. The decrease in the emission intensity on addition of the complexes is shown in Fig. 2 and the tentative binding order from the slope of the plot is: [Cu(Ph-tsc) (dppz)] (3) > [Cu(phen)2 (H2 O)]2þ > [Cu(Ph-tsc)(dpq)] (2) > [Cu(Ph-tsc)(phen)] (1) > [Cu(Me-tsc)(phen)] (4). Earlier studies on bis-phen copper complexes have shown that this complex binds to DNA either by partial intercalation or binding of one phen ligand to the minor groove, while the other phen making favorable contacts within the groove [3,17,48]. The ancillary ligand plays an important role in DNA groove binding or intercalation. The observed results suggest the greater binding of the dppz complex in comparison to the dpq or phen species. This could be related to its extended aromatic ring or due to its different groove selectivity. We have earlier reported the binding properties of a series of ternary copper(II) complexes having tris(3-phenylpyrazolyl)borate and planar heterocyclic base [44]. It has been observed that while phen and dpq bind at the minor groove, dppz prefers major groove binding. The greater
2320
A.M. Thomas et al. / Inorganica Chimica Acta 357 (2004) 2315–2323
Fig. 2. Effect of addition of [Cu(Ph-tsc)B] [B ¼ phen (1, j); dpq (2, ); dppz (3, N)], [Cu(Me-tsc)(phen)] (4, }) and [Cu(phen)2 (H2 O)](ClO4 )2 (s) to the emission intensity of 125 lM CT DNA-bound ethidium bromide (12.5 lM) in a 5 mM Tris–HCl/50 mM NaCl buffer (pH ¼ 7.2) at 25 °C (*, emission intensity of ethidium bromide in the absence of DNA).
binding of 1 to CT DNA than 4 indicates more favorable contact of Ph-tsc with DNA than its Me-tsc analogue. The oxidative DNA cleavage activity of the complexes was studied by gel electrophoresis using supercoiled (SC) pUC19 DNA in Tris–HCl buffer (pH, 7.2) in the presence of a reducing agent like mercaptopropionic acid (MPA). The complexes show significant cleavage of DNA in dark in the presence of MPA (Table 4, Fig. 3). Control experiments by treating DNA with only MPA or 1 do not show any apparent cleavage of DNA. Comparative studies reveal that the ‘‘chemical nuclease’’ activity of 1–4 is relatively less than that of the bis-(phen)copper(II) species. The observed DNA cleavage order 3 > 2 > 1 > 4 resembles well to their propensity of binding to DNA. Although the true identity of the active species in the oxidative DNA cleavage involving bis(phen)copper and its related complexes is not known, it has been proposed that the Table 4 Cleavage of SC pUC19 DNA (500 ng) by 1–4 (30 lM) in the presence of mercaptopropionic acid (MPA, 50 mM) in the absence of light Serial numbers
1 2 3 4 5 6 7 8
Reaction condition
DNA control DNA + 1 + MPA DNA + 2 + MPA DNA + 3 + MPA DNA + 4 + MPA DNA + bis(phen)Cu(II) + MPA DNA + Distamycin + 2 + MPA DNA + Distamycin + 3 + MPA
% cleavage Form I
Form II
96 59 30 16 70 3
4 41 70 84 30 97
90
10
32
68
oxidative attack at the sugar moiety involves either free hydroxyl radical or a copper bound oxo or hydroxo species [6]. We have carried out mechanistic investigations using DMSO as a scavenger for the hydroxyl radical. The complexes indeed show complete inhibition of cleavage in the presence of DMSO, indicating a reaction pathway that could be similar to the one proposed by Sigman and coworkers [3] for the oxidative cleavage of DNA by bis(phen)copper complex. The groove binding preferences of the complexes are studied using minor groove binder distamycin. While phen and dpq complexes display significant inhibition of cleavage in the presence of distamycin, the dppz complex exhibits only minor inhibition (Table 4). This suggests the minor groove preference of the phen and dpq complexes, while the dppz complex binds at the major groove [44]. 3.4. Photo-induced DNA cleavage The light-induced supercoiled (SC) DNA cleavage activity of the complexes has been studied using UV radiation of wavelength 312 nm and green light of 532 nm (Table 5, Fig. 4). When exposed to UV light for 5 min, the 50 lM solutions of the complexes show 80% SC DNA cleavage for the dpq and dppz complexes, while the phen complex 1 displays moderate cleavage (50%). Among the phen complexes, the Me-tsc species 4 shows significantly less activity than its Ph-tsc analogue. In the ternary structure, the heterocyclic base acts as a DNA binder. The thiosemicarbazone base and the heterocyclic ligands dpq and dppz with their quinoxaline and phenazine moiety, respectively, act as photosensitizer. The observed lower cleavage activity of the Me-tsc complex 4 could be related to its poor binding ability to DNA. The mechanistic aspects of the photo-cleavage reaction have been probed for the major groove binder dppz complex 3 using different reagents. Addition of DMSO, ethanol, mannitol or sodium formate does not inhibit the cleavage activity of the complex, indicating non-involvement of the hydroxyl radical in the photo cleavage reaction. The complex is cleavage inactive under argon atmosphere. Under aerobic conditions, sodium azide shows significant inhibition. The cleavage activity enhances in D2 O. The results suggest the involvement of singlet oxygen (1 O2 ) as a reactive species. Control experiments show that the thiosemicarbazones, phen and dppz, alone are nuclease inactive on photo irradiation at 312 nm. The dpq ligand with its quinoxaline moiety, however, shows significant cleavage at UV radiation. In a recent publication, Toshima and coworkers have shown that quinoxalines, similar to those present in the antitumor antibiotics like triostin and echinomycin, are cleavage active at 365 nm without any external additives by involving C@N bond
A.M. Thomas et al. / Inorganica Chimica Acta 357 (2004) 2315–2323
2321
Fig. 3. Gel diagram showing the oxidative DNA cleavage activity of the complexes 1–4 along with [Cu(phen)2 (H2 O)]2þ in the presence of mercaptopropionic acid (MPA) under different reaction conditions using pUC19 DNA (500 ng) in DMF–Tris–HCl buffer medium (pH 7.2). Lane 1, DNA control; Lane 2, DNA + MPA; Lane 3, DNA + 1; Lane 4, DNA + 1 + MPA; Lane 5, DNA + 2 + MPA; Lane 6, DNA + 3 + MPA; Lane 7, DNA + 4 + MPA; Lane 8, DNA + [Cu(phen)2 (H2 O)]2þ + MPA, Lane 9, DNA + distamycin + 1 + MPA; Lane 10, DNA + distamycin + 2 + MPA; Lane 11, DNA + distamycin + 3 + MPA; Lane 12, DNA + distamycin + 4 + MPA; Lane 13, DNA + distamycin + [Cu(phen)2 (H2 O)]2þ + MPA; Lane 14, DNA + DMSO + 1 + MPA. (complex, 30 lM; MPA, 50 mM; distamycin, 100 lM; DMSO, 4 lL).
Table 5 Photo-cleavage data of SC pUC19 DNA (500 ng) by 1–4 on UV and visible light irradiationa Reaction condition
DNA control DNA + 1 DNA + 2 DNA + 3 DNA + 4 DNA control DNA + dpqb DNA + 1 DNA + 2 DNA + 3 a b
[Complex]/lM
50 50 50 50
200 200 200
k/nm
312 312 312 312 312 532 532 532 532 532
t/min
5 5 5 5 5 30 30 30 30 30
% cleavage Form I
Form II
94 50 25 20 80 90 91 87 49 47
6 50 75 80 20 10 9 13 51 53
t, exposure time. 200 lM.
Fig. 4. (a). Gel diagram showing the photo-cleavage data of SC pUC19 DNA (500 ng) by 1–4 (50 lM for UV and 200 lM for visible radiation) on irradiation with UV light (312 nm, 5 min exposure, Lanes 1–9) and visible light (532 nm, 30 min exposure, Lanes 10–14). Lane 1, DNA control; Lane 2, DNA + Ph-H2 tsc; Lane 3, DNA + dpq; Lane 4, DNA + dppz; Lane 5, DNA + [Cu(phen)2 (H2 O)]2þ ; Lane 6, DNA + 1, Lane 7, DNA + 2; Lane 8, DNA + 3; Lane 9, DNA + 4; Lane 10, DNA control; Lane 11, DNA + dpq; Lane 12, DNA + 1; Lane 13, DNA + 2; Lane 14, DNA + 3 Ligand concentration for control experiments: 50 lM for UV and 200 lM for visible radiation. (b) Gel diagram displaying the cleavage data of SC pUC19 DNA (500 ng) by complex 3 (70 lM) in the presence of various hydroxyl radical scavengers at 312 nm with 5 min exposure time: Lane 1, DNA control; Lane 2, DNA + ethanol (5 lL); Lane 3, DNA + mannitol (100 mM); Lane 4, DNA + sodium formate (100 mM); Lane 5, DNA + DMSO (4 lL); Lane 6, DNA + 3; Lane 7, DNA + ethanol + 3; Lane 8, DNA + mannitol + 3; Lane 9, DNA + sodium formate + 3; Lane 10, DNA + DMSO + 3. (c) Gel diagram displaying the cleavage data of SC pUC19 DNA (500 ng) by complex 3 (90 M) in the presence of various singlet oxygen quenchers at 312 nm with 5 min exposure time: Lane 1, DNA control; Lane 2, DNA + sodium azide (10 mM); Lane 3, DNA + D2 O (14 lL); Lane 4, DNA + 3; Lane 5, DNA + sodium azide + 3; Lane 6, DNA + 3 under argon; Lane 7, DNA + D2 O (14 lL) + 3.
that could generate the photo-excited 3 (n–p ) and/or 3 (p–p ) states causing DNA damage by H-abstraction and/or electron transfer pathways(s) [49]. Control experiments using mono-dpq or mono-dppz copper(II) complex show 90 and 72% cleavage of SC DNA at 312 nm under similar conditions [34]. The reduced activity of the dppz complex than its dpq analogue in
the absence of the thiosemicarbazone ligand could be due to the phenyl ring of the phenazine moiety in dppz reducing the lifetime of its triplet 3 (n–p ) and/or 3 (p–p ) state. It appears from the cleavage data that complexes 2 and 3 are undergoing dual photosensitizing effect, involving the thiosemicarbzone and the heterocyclic ligand. The marginal enhancement of %SC
2322
A.M. Thomas et al. / Inorganica Chimica Acta 357 (2004) 2315–2323
DNA cleavage observed for the dppz complex than its dpq analogue may be attributed to the effect of their different groove selectivity. The cleavage data also suggest that the conjugation present in the thiosemicarbazone ligand makes this ligand inefficient possibly by reducing the triplet state lifetime of the sulfur ligand in an enolic form. As a consequence, the photosensitizing effect is more prominent for the quinoxaline and phenazine moieties than that of the sulfur ligand. We have recently reported the photonuclease activity of mono-(phen)copper(II) complex containing NSO-donor Schiff base, 2-(methylthio)ethylsalicylaldimine [22]. Absence of any conjugation between the thio group and the imine moiety enhances the photosensitizing effect. We have probed the photo-nuclease activity of the dpq and dppz complexes using a visible light source of 532 nm. Moderate cleavage is observed for 2 and 3 on irradiation at this wavelength (Table 5, Fig. 4). Control experiments show that the ligands alone are cleavage inactive at this wavelength. The results indicate the possible involvement of the copper(II) center in the formation of singlet oxygen, involving initial photosensitization process of the sulfur ligand as well as the heterocyclic base. The moderate cleavage activity could also be due to the possibility of the sulfur atom of the thiosemicarbazone ligand undergoing oxidation in the presence of singlet oxygen [50]. Mechanistic studies indicate the formation of singlet oxygen as the active species cleaving DNA as a major pathway. The alternate pathway leading to the formation of hydroxyl or copper-oxo species, by involving the photo-activated ligand reducing the copper from +2 to +1 oxidation state in a process similar to that proposed by Sigman and coworkers for the ‘‘chemical nuclease’’ reaction of the bis-phen copper complex, seems to be less probable [3,17].
4. Conclusions New ternary copper(II) complexes having thiosemicarbazones as photosensitizer and phenanthroline bases as DNA binder are prepared, structurally characterized, and their DNA cleavage properties studied. The dpq and dppz complexes show significant cleavage of SC DNA on irradiation with green light in the absence of any additives. The dpq and dppz complexes also show dual photosensitizing effect by involving the thiosemicarbazone ligand and the quinoxaline or phenazine moiety of the heterocyclic base. The dpq and phen complexes are minor groove binder, while the dppz complex binds at the major groove of DNA. Mechanistic studies reveal possible involvement of hydroxyl radical and/or copper bound oxo/hydroxo species in the oxidative cleavage of DNA in the presence of merca-
ptopropionic acid. The photocleavage reactions under UV or visible light irradiation involve singlet oxygen as the DNA cleaving agent. The present results are of significance, considering the pharmacological and antitumor activity of thiosemicarbazones and the antitumor activities of quinoxaline antibiotics. Our observation of the copper(II) center assisting the photo-cleavage of DNA is of significance as the same metal center, in contrast, is known to reduce the cleavage activity of porphyrin-copper(II) complexes by significantly reducing the 3 (p–p ) state lifetime of the porphyrin bases [23]. We have observed a significant effect of conjugation in the sulfur containing Schiff base in reducing the cleavage efficiency. The results are of importance in designing copper-based complexes containing sulfur containing ligands for PDT applications.
5. Supplementary material Crystallographic data (excluding structure factors) for the structure have been deposited with the Cambridge Crystallographic Data Center, CCDC reference no. 215179. Copies of this information may be obtained free of charge from The Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: (internat.) +441223-336-033; e-mail: [email protected] or http:// www.ccdc.cam.ac.uk).
Acknowledgements We thank the Council of Scientific and Industrial Research (CSIR), New Delhi, and the Department of Science and Technology (DST), Government of India, for the financial support; DST for the CCD diffractometer facility; the Alexander von Humboldt Foundation, Germany, for donation of an electroanalytical system; and the Convener, Bioinformatics Center, Indian Institute of Science, Bangalore, for database search.
References [1] (a) B. Meunier, Chem. Rev. 92 (1992) 1411; (b) G. Pratviel, J. Bernadou, B. Meunier, Angew. Chem., Int. Ed, Engl. 34 (1995) 746; (c) M. Pitie, J.D.V. Horn, D. Brion, C.J. Burrows, B. Meunier, Bioconjugate Chem. (2000) 892. [2] K.E. Erkkila, D.T. Odom, J.K. Barton, Chem. Rev. 99 (1999) 2777. [3] (a) D.S. Sigman, T.W. Bruce, A. Mazumder, C.L. Sutton, Acc. Chem. Res. 26 (1993) 98; (b) D.S. Sigman, A. Mazumder, D.M. Perrin, Chem. Rev. 93 (1993) 2295. [4] S.J. Lippard, Chem. Rev. 99 (1999) 2467.
A.M. Thomas et al. / Inorganica Chimica Acta 357 (2004) 2315–2323 [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
[15]
[16]
[17]
[18] [19]
[20]
[21] [22]
[23]
[24]
J. Reedijk, J. Inorg. Biochem. 86 (2001) 89. C.J. Burrows, J.G. Muller, Chem. Rev. 98 (1998) 1109. B. Armitage, Chem Rev. 98 (1998) 1171. W.K. Pogozelski, T.D. Tullius, Chem. Rev. 98 (1998) 1089. D.R. Mc Millin, K.M. Mc Nett, Chem. Rev. 98 (1998) 1201. S.J. Lippard, J.M. Berg, Prinicples of Bioinorganic Chemistry, University Science Books, CA, 1994. R. Ackroyd, C. Kelty, N. Brown, M. Reed, Photochem. Photobiol. 74 (2001) 656. B.W. Henderson, T.J. Dougherty, Photochem. Photobiol. 55 (1992) 145. M.C. De Rose, R.J. Crutchley, Coord. Chem. Rev. 233–234 (2002) 351. (a) E.D. Sternberg, D. Dolphin, C. Br€ uckner, Tetrahedron 54 (1998) 4151; (b) J.L. Sessler, G. Hemmi, T.D. Mody, T. Murai, A. Burrell, S.W. Young, Acc. Chem. Res. 27 (1994) 43; (c) H. Ali, J.E. Van Lier, Chem. Rev. 99 (1999) 2379. B.W. Henderson, T.M. Busch, L.A. Vaughan, N.P. Frawley, D. Babich, T.A. Sosa, J.D. Zollo, A.S. Dee, M.T. Cooper, D.A. Bellnier, W.R. Greco, A.R. Oseroff, Cancer Res. 60 (2000) 525. (a) O. Baudoin, M.-P. Teulade-Fichou, J.-P. Vigneron, J.-M. Lehn, Chem. Commun. (1998) 2349; (b) M. Gonzalez-Alvarez, G. Alzuet, J. Borras, M. Pitie, B. Meunier, J. Biol. Inorg. Chem. 8 (2003) 644; (c) J.H Kim, S.H. Kim, Chem. Lett. 32 (2003) 490; (d) A. Garca-Raso, J.J. Fiol, B. Adrover, V. Moreno, I. Mata, E. Espinosa, E. Molins, J. Inorg. Biochem. 95 (2003) 77; (e) L.-P. Lu, M.-L. Zhu, P. Yang, J. Inorg. Biochem. 95 (2003) 31; (f) J.A. Cowan, Chem. Commun. (2001) 1490. (a) D.S. Sigman, Biochemistry 29 (1990) 9097; (b) T.B. Thederahn, M.D. Kuwabara, T.A. Larsen, D.S. Sigman, J. Am. Chem. Soc. 111 (1989) 4941; (c) O. Zelenko, J. Gallagher, D.S. Sigman, Angew. Chem., Int. Ed. Engl. 36 (1997) 2776; (d) M.M. Meijler, O. Zelenko, D.S. Sigman, J. Am. Chem. Soc. 119 (1997) 1135. E.L. Hegg, J.N. Burstyn, Coord. Chem. Rev. 173 (1998) 133. (a) D.K. Chand, H.-J. Schneider, A. Bencini, A. Bianchi, C. Giorgi, S. Ciattini, B. Valtancoli, Chem. Eur. J. 6 (2000) 4001; (b) R. Hettich, H.-J. Schneider, J. Am. Chem. Soc. 119 (1997) 5638. (a) J.A. Cowan, Chem. Rev. 98 (1998) 1067; (b) F.H. Westheimer, Science 235 (1987) 1173; (c) D.E. Wilcox, Chem. Rev. 96 (1996) 2435; (d) M. Komiyama, J. Sumaoka, Curr. Opin. Chem. Biol. 2 (1998) 751. H.J. Eppley, S.M. Lato, A.D. Ellington, J.M. Zaleski, Chem. Commun. (1999) 2405. (a) S. Dhar, A.R. Chakravarty, Inorg. Chem. 42 (2003) 2483; (b) A.K. Patra, S. Dhar, A.R. Chakravarty, Chem. Commun. (2003) 1562; (c) S. Dhar, D. Senapati, P.K. Das, P. Chattopadhyay, M. Nethaji, A.R. Chakravarty, J. Am. Chem. Soc. 125 (2003) 12118; (d) S. Dhar, D. Senapati, P.A.N. Reddy, P.K. Das, A.R. Chakravarty, Chem. Commun. (2003) 2452. (a) D. Praseuth, A. Gaudemer, J.-B. Verlhac, I. Kraljic, I. Sissoeff, E. Guille, Photochem. Photobiol. 44 (1986) 717; (b) S. Somer, C. Rimington, J. Mohan, FEBS Lett. 172 (1984) 267. M. Baldini, M.B. Ferrari, F. Bisceglie, G. Pelosi, S. Pinelli, P. Tarasconi, Inorg. Chem. 42 (2003) 2049.
2323
[25] Q. Xuhong, H. Tian-Bao, W. Dong-zhi, Z. Dong-Hui, F. Mingcai, Y. Wei, J. Chem. Soc., Perkin Trans. 2 (2000) 715. [26] A. Jakobs, J. Piette, J. Photochem. Photobiol. B. Biol (1994) 2219. [27] A. Jakobs, J. Piette, J. Med. Chem. 38 (1995) 869. [28] (a) S. Padhye, S.B. Kauffman, Coord. Chem. Rev. 63 (1985) 127; (b) D.X. West, A.E. Liberta, S.B. Padhye, R.C. Chikate, P.B. Sonawane, A.S. Kumbhar, R.G. Yerande, Coord. Chem. Rev. 123 (1993) 49. [29] R.W. Byrnes, M. Mohan, W.E. Antholine, R.X. Xu, D.H. Petering, Biochemistry 29 (1990) 7046. [30] (a) Z. Afrasiabi, E. Sinn, S. Padhye, S. Dutta, S. Padhye, C. Newton, C.E. Anson, A.K. Powell, J. Inorg. Biochem. 95 (2003) 306; (b) A.I. Matesanz, J.M. Perez, P. Navarro, J.M. Moreno, E. Colacio, P. Souza, J. Inorg. Biochem. 76 (1999) 29. [31] D.K. Demertzi, P.N. Yadav, M.A. Demertzis, M. Coluccia, J. Inorg, Biochem. 78 (2000) 347. [32] Z. Lakovidou, A. Papgeorgiou, M.A. Demertzis, E. Mioglou, D. Mourelatos, A. Kotsis, P.N. Yadav, D.K. Demertzi, Anti Cancer Drugs 12 (2001) 65. [33] (a) K.H. Reddy, P.S. Reddy, P.R. Babu, Trans. Met. Chem. 25 (2000) 154; (b) K.H. Reddy, P.S. Reddy, P.R. Babu, J. Inorg. Biochem. 77 (1999) 169. [34] A.M. Thomas, M. Nethaji, S. Mahadevan, A.R. Chakravarty, J. Inorg. Biochem. 94 (2003) 171. [35] (a) H. Nakai, Bull. Chem. Soc. Jpn. 44 (1971) 2412; (b) J. Foley, D. Kennefick, D. Phelan, S. Tyagi, B. Hathaway, J. Chem. Soc. Dalton Trans. (1983) 2333. [36] D.L. Klayman, J.F. Brtosevich, T.S. Griffin, C.J. Mason, J.P. Scovill, J. Med. Chem. 22 (1997) 855. [37] J. Bernadou, G. Pratviel, F. Bennis, M. Girardet, B. Meunier, Biochemistry 28 (1989) 7268. [38] (a) E. Amouyal, A. Homsi, J.-C. Chambron, J.-P. Sauvage, J. Chem. Soc., Dalton Trans. (1990) 1841; (b) J.E. Dickenson, L.A. Summers, Aust. J. Chem. 23 (1970) 1023. [39] M.E. Reichmann, S.A. Rice, C.A. Thomas, P. Doty, J. Am. Chem. Soc. 76 (1954) 3047. [40] R. Blessing, Acta Crystallogr. Sect A. 51 (1995) 33. [41] G.M. Sheldrick, S H E L X -97, Programs for Crystal Structure Solution and Refinement, University of G€ ottingen, Germany, 1997. [42] C.K. Johnson, ORTEP, Report ORNL – 5138, Oak Ridge National Laboratory, Oak Ridge, TN, 1976. [43] A.D. Naik, P.A.N. Reddy, M. Nethaji, A.R. Chakravarty, Inorg. Chim. Acta 349 (2003) 149. [44] S. Dhar, P.A.N. Reddy, M. Nethaji, S. Mahadevan, M.K. Saha, A.R. Chakravarty, Inorg. Chem. 41 (2002) 3469. [45] A.W. Addison, T.N. Rao, J. Reedijk, J.V. Riju, G.C. Verschoor, J. Chem. Soc., Dalton Trans. (1984) 1349. [46] L.C. Zheng, W. Jigui, W. Liufang, R. Min, J. Naiyong, G. Jie, J. Inorg. Biochem. 73 (1999) 195. [47] M.J. Waring, J. Mol. Biol. 13 (1965) 269. [48] (a) B.C. Bales, M. Pitie, B. Meunier, M.M. Greenberg, J. Am. Chem. Soc. 124 (2002) 9062; (b) J.M. Veal, K. Merchant, R.L. Rill, Nucleic Acids Res. 19 (1991) 3383. [49] K. Toshima, R. Takano, T. Ozawa, S. Matsumura, Chem. Commun. (2002) 212. [50] F. Champloy, N. Benali-Cherif, P. Bruno, I. Blain, M. Pierrot, M. Reglier, Inorg. Chem. 37 (1998) 3910.
Synthesis, crystal structure and photo-induced DNA cleavage activity of ternary copper(II)-thiosemicarbazone complexes having heterocyclic bases q Anitha M. Thomas, Anil D. Naik, Munirathinam Nethaji, Akhil R. Chakravarty
*
Department of Inorganic and Physical Chemistry, Indian Institute of Science, Sir C.V. Raman Avenue, Bangalore 560012, India Received 1 October 2003; accepted 31 January 2004 Available online 21 February 2004
Abstract New ternary copper(II) complexes of formulations [Cu(Ph-tsc)B] (B ¼ 1,10-phenanthroline, phen (1); dipyridoquinoxaline, dpq (2); dipyridophenazine, dppz (3); Ph-H2 tsc, salicylaldehyde-N(4)-phenylthiosemicarbazone) and [Cu(Me-tsc)(phen)] (4, Me-H2 tsc, salicylaldehyde-N(4)-methylthiosemicarbazone) are prepared, and their DNA binding and cleavage properties studied. Complex 1 has been characterized by single crystal X-ray crystallography. The molecular structure shows a distorted square pyramidal (4 + 1) geometry of the complex with the dianionic NSO-donor N(4)-phenyl-substituted thiosemicarbazone binding at the basal plane and the NN-donor planar heterocyclic base (phen) displaying axial–equatorial coordination. The one-electron paramagnetic complexes exhibit axial EPR spectra and show a d–d band near 580 nm for the phen and near 720 nm for the dpq, dppz complexes in their electronic spectra in DMF. The complexes show quasireversible cyclic voltammetric response near 0.08 V vs. SCE in DMF–0.1 M TBAP assignable to the Cu(II)/Cu(I) couple. The Ph-tsc complexes display good binding propensity to calf thymus (CT) DNA. They also show oxidative cleavage of supercoiled (SC) pUC19 DNA in dark under aerobic condition in the presence of mercaptopropionic acid. The complexes exhibit light-induced DNA cleavage activity at 312 and 532 nm. Mechanistic investigations reveal DNA minor groove binding for the phen and dpq complexes, and major groove binding for the dppz species. The complexes are cleavage inactive under argon atmosphere. In the ternary structure, the thiosemicarbazones, dpq and dppz act as photosensitizers, while the planar heterocyclic bases are binder to DNA. The mechanistic pathways involved and the role of metal in the DNA cleavage reactions are discussed. Ó 2004 Elsevier B.V. All rights reserved. Keywords: Ternary copper(II) complex; Thiosemicarbazones; Heterocyclic bases; DNA Photocleavage; Crystal structure
1. Introduction Designing transition metal complexes, that can bind and cleave DNA under physiological conditions, is of importance in the development of diagnostic agents for medicinal applications [1–10]. The DNA cleavage reactions generally proceed via three major pathways, viz. oxidative strand cleavage by abstraction of sugar hydrogen atom(s), hydrolytic cleavage involving the phosphate group, and by base oxidations primarily directed at the guanine base. Among different methodolq Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ica.2004.01.031. * Corresponding author. Tel.: +918022932533; fax: +918023600683. E-mail address: [email protected] (A.R. Chakravarty).
0020-1693/$ - see front matter Ó 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ica.2004.01.031
ogies adopted for inducing DNA cleavage by synthetic nucleases, the one based on photo-irradiation has received current attention with the advent of photodynamic therapy (PDT) of cancer [11–15]. The PDT process generally requires a photosensitizer (drug), a visible light source (preferably red light), and oxygen. The drug on photo-excitation is promoted to its excited singlet state. It then populates triplet states due to rapid intersystem crossing followed by activation of triplet oxygen to its singlet state. Porphyrin derivatives have largely been used as photosensitizers for medicinal applications in PDT [14,15]. The present work stems from our continued interest to design copper-based transition metal complexes cleaving DNA on photo-irradiation. Although there are reports [16–20] of copper complexes cleaving DNA hydrolytically or in an oxidative manner
2316
A.M. Thomas et al. / Inorganica Chimica Acta 357 (2004) 2315–2323
in the presence of a reducing agent, the chemistry of copper complexes showing visible light-induced photocleavage is virtually unexplored [21,22]. We have reported that non-porphyrinic copper(II) complexes can cause significant cleavage of supercoiled DNA in the presence of suitable photosensitizer(s) in a ternary structure [22]. Our observation of copper(II)-assisted visible light-induced DNA photo-cleavage in a ternary complex is important as the copper(II)–porphyrin complexes, in contrast, are known to be inefficient DNA photo-cleaver due to significant effect of the metal center in quenching the porphyrin 3 (p–p ) state life time [23]. The utility of thiosemicarbazone-copper(II) complexes in DNA binding and in vitro studies has recently been reported by Baldini and coworkers [24]. We have probed the DNA binding and photocleavage properties of a series of ternary copper(II) complexes [Cu(Ph-tsc)B] (B ¼ phen (1); dpq, (2); dppz (3)) and [Cu(Metsc)(phen)] (4) (Chart 1), containing N(4)-mono-substituted thiosemicarbazones and planar phenanthroline bases. Our choice for the sulfur-containing thiosemicarbazones is based on the fact that thio or thione moieties are known to show efficient intersystem crossing to the triplet state on photo-irradiation [22,25–27]. Such an excited state with a longer lifetime can either cause damage to DNA directly or can activate oxygen from its stable triplet (3 O2 ) to the cytotoxic singlet state (1 O2 ). The choice of thiosemicarbazones is based on their significant pharmacological properties. Besides, several thiosemicarbazone transition metal complexes are known to exhibit potential antitumor activities [28–30]. It has been proposed that the cellular oxidative chemistry, involving copper thiosemicarbazones in the absence of photo-irradiation, is similar to those of iron(II) bleomycin or copper(I) phenanthroline complexes [31– 33]. In our scheme of work, we have used thiosemicarbazones (Ph-tsc, Me-tsc) as potential photosensitizers in ternary copper(II) complexes in which the role of heterocyclic bases, with extended planar aromatic ring(s),
Chart 1.
viz. 1,10-phenanthroline (phen), dipyridoquinoxaline (dpq) or dipyridophenazine (dppz), is to primarily bind to double-stranded DNA. In addition, the dpq and dppz ligands can also act as photosensitizer with their respective quinoxaline and phenazine moieties. Significant results of the present study include the observation of efficient DNA cleavage by the dpq and dppz complexes on photo-irradiation at green light (k, 532 nm) and different DNA-groove selectivity of the complexes.
2. Experimental 2.1. Chemical reagents All reagents and chemicals of analytical grade were purchased from commercial sources. Solvents used for spectral and electrochemical measurements were purified by standard procedures. Salicylaldehyde, N(4)-phenylthiosemicarbazide, and N(4)-methylthiosemicarbazide were obtained from Aldrich (USA). Copper(II) acetate hydrate and 1,10-phenanthroline (phen) were purchased from SD Fine Chemicals, Mumbai. Calf thymus (CT) DNA and supercoiled (SC) pUC19 DNA (cesium chloride purified) were from Bangalore Genie (India). Agarose (molecular biology grade), ethidium bromide (EB) and distamycin were obtained from Sigma (USA). Tris–HCl buffer solution was prepared using deionized, sonicated triple distilled water. Complexes [Cu(dpq) (NO3 )2 ], [Cu(dppz)(NO3 )2 ] and [Cu(phen)2 (H2 O)] (ClO4 )2 , used for control experiments, were prepared by reported methods [34,35]. The thiosemicarbazones (PhH2 tsc and Me-H2 tsc) were prepared by a literature method [36]. The heterocyclic bases, dipyrido[3,2-d:20 ,30 f]quinoxaline (dpq) and dipyrido[3,2-a:20 ,30 -c]phenazine (dppz), were prepared using 1,10-phenanthroline as a precursor by literature procedures [37,38]. 2.2. Measurements The elemental analytical data were obtained from a Heraeus CHN–O rapid instrument. The infrared, electronic, and EPR spectral data were recorded using Bruker Equinox 55, Hitachi U-3400, and Varian E-109 X-band spectrometers, respectively. Magnetic susceptibility data at room temperature were obtained from a George Associates Inc. (Berkeley, CA) Lewis-coil force magnetometer. Hg[Co(NCS)4 ] was used as a standard. Cyclic voltammetric measurements were done at 25 °C on a EG&G PAR Model 253 Versa Stat potentiostat/ galvanostat with electroanalytical software 270 using a three electrode setup consisting of a glassy carbon working, platinum wire auxiliary, and a saturated calomel reference (SCE) electrode. Tetrabutylammonium perchlorate (TBAP, 0.1 M) was used as supporting electrolyte. The Fe(III)/Fe(II) couple of ferrocene as a
A.M. Thomas et al. / Inorganica Chimica Acta 357 (2004) 2315–2323
standard was observed at 0.4 V vs. SCE in DMF–0.1 M TBAP. 2.3. Synthesis of the compounds 2.3.1. [Cu(Ph-tsc)(phen)] (1) and [Cu(Me-tsc) (phen)] (4) The complexes were prepared by a general method, in which copper(II) acetate hydrate (0.2 g, 0.5 mmol) was reacted with 1,10-phenanthroline (phen, 0.2 g, 1.0 mmol) and the thiosemicarbazone (Ph-H2 tsc, 0.27 g; Me-H2 tsc, 0.21 g; 1.0 mmol) in 25 mL MeOH. The solution was refluxed for 3 h, followed by cooling to an ambient temperature. The solid thus obtained was washed with cold methanol and dried under vacuum over P4 O10 (Yield: 65%). Anal. Calc. for C26 H19 N5 OSCu (1): C, 60.86; H, 3.73; N, 13.64. Found: C, 60.72; H, 3.94; N, 13.73%. IR (KBr phase, cm1 ): 3398w, 3056w, 1600s, 1534s, 1495s, 1421s, 1314m, 840m, 736m (s, strong; m, medium; w, weak). Anal. Calc. for C21 H17 N5 OSCu (4): C, 55.93; H, 3.80; N, 15.53. Found: C, 55.76, H, 3.57; N, 15.63%. IR (KBr disc, cm1 ): 3231w, 3050w, 1600s, 1513s, 1448m, 1403m, 1340m, 1198m, 849m, 724m. 2.3.2. [Cu(Ph-tsc)(dpq)] (2) and [Cu(Ph-tsc)(dppz)] (3) The complexes were synthesized using a general procedure in which a hot methanol solution (60 °C, 25 mL) of the thiosemicarbazone ligand (Ph-H2 tsc, 0.27 g, 1.0 mmol) was initially reacted with copper(II) acetate hydrate (0.2 g, 0.5 mmol) taken in methanol (5 mL) under magnetic stirring for 30 min. The solution was cooled to an ambient temperature and the heterocyclic base (1.0 mmol; dpq, 0.23 g; dppz, 0.28 g) dissolved in CHCl3 (20 mL) was added to the solution that was stirred for a further period of 2 h. Diethyl ether was added to the solution to precipitate the solid which was isolated, washed with CHCl3 , and finally dried in vacuo over P4 O10 . Yield: 60%. Anal. Calc. for C28 H19 N7 OSCu (2): C, 59.51; H, 3.38; N, 17.35. Found: C, 59.67; H, 3.28; N, 17.21%. IR (KBr phase, cm1 ): 3425w, 3065w, 1600m, 1573m, 1483m, 1385s, 1078m, 810m, 736m. Anal. Calc. for C32 H21 N7 OSCu (3): C, 62.47; H, 3.44; N, 15.93. Found: 62.54; H, 3.57; N, 16.15%. IR (KBr disc, cm1 ): 3446w, 3076w, 1600s, 1492m, 1373m, 1098m, 813m, 754m, 730m. 2.4. DNA binding and cleavage experiments The concentration of calf thymus (CT) DNA was determined from the absorption intensity at 260 nm with a molar extinction coefficient value of 6600 M1 cm1 in Tris–HCl/NaCl buffer (pH 7.2) [39]. In a typical binding experiment, a 2.0 mL solution of ethidium bromide (EB) was added to a volume of 100 lL of 125 lM CT DNA solution to get the maximum fluorescence intensity of
2317
EB (excitation at 510 nm; emission at 601 nm). Aliquots of 10 mM solution of the complex in DMF were added to the EB bound CT DNA solution and the fluorescence intensity had been measured after each addition until a 50% reduction of the intensity was observed. The fluorescence intensities were plotted against the complex concentration to get a slope that gave the relative measure of the extent of binding of the complex to the CT DNA. A control experiment was also done with the EB in the absence of CT DNA and the observed intensities were plotted against the complex concentration. DNA cleavage activity of the complexes was monitored by agarose gel electrophoresis in the presence or absence of a reducing agent under dark or illuminated conditions. In a typical ‘‘chemical nuclease’’ activity study, supercoiled (SC) pUC19 DNA (6 lL, 500 ng) in Tris–HCl buffer (50 mM, pH 7.2) containing 50 mM NaCl (2 lL) was treated with the ternary copper(II) complex (2 lL, 30 lM) and mercaptopropionic acid (2 lL, 50 mM) followed by dilution with the buffer to a total volume of 20 lL in a dark chamber. The samples were then incubated for 1 h at 37 °C, added loading buffer containing 25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol (3 lL) and loaded on 0.8% agarose gel containing 1.0 lg/mL ethidium bromide. Electrophoresis was carried out at 40 V for 2 h in Tris-acetateEDTA (TAE) buffer. Bands were visualized by UV light and photographed followed by the estimation of the intensity of the DNA bands using UVITEC Gel Documentation system. Due correction was made for the low level of NC (nicked circular) DNA present in the original sample and the low affinity of ethidium bromide binding to SC DNA in comparison to its NC form [37]. Inhibition reactions were carried out under dark condition by prior incubation of the SC DNA with DMSO (5 lL) or distamycin (2 lL, 100 lM) for 10 min before the addition of the ternary complex and the reducing agent (MPA), and then subjected for further incubation and gel electrophoresis using the above procedures. The photo-induced pUC19 DNA cleavage studies were done using monochromatic UV (k ¼ 312 nm, 96 W (total wattage) transilluminator UVITEC make) and visible (k ¼ 532 nm, mercury lamp of 125 W of commercial source) light in the absence of MPA. For mechanistic investigations, the inhibition reactions were carried out at 312 nm using different reagents like sodium azide (10 mM), DMSO (4 lL), mannitol (100 mM) and sodium formate (100 mM). For the D2 O experiment, this solvent was used for dilution to 18 lL. The photo-cleavage experiment was also done under an argon atmosphere. 2.5. X-ray crystallography Greenish-brown crystals of 1 H2 O were grown by a diffusion technique in which diethyl ether was layered above the complex solution in MeOH–CHCl3 mixture
2318
A.M. Thomas et al. / Inorganica Chimica Acta 357 (2004) 2315–2323
Table 1 Selected crystallographic data for [Cu(Ph-tsc)(phen)] (1 H2 O) 1 H2 O Empirical formula Crystal size Crystal color Crystal morphology Crystal system Space group Unit cell dimensions a (A) b (A) c (A)
C26 H21 CuN5 O2 S 0.2 0.14 0.1 greenish-brown rectangular monoclinic Cc 17.603(4) 11.435(2) 12.344(3) 107.089(3) 2375.1(8) 4 1.485 1.042 1092 0.71073 293(2) 4203 3935 316 0.0442 0.1106 0.0467 0.1125 1.021 0.000 0.414 and )0.264
b (°) 3 ) V (A Z Dcalc (g cm3 ) l(Mo) (mm1 ) F ð0 0 0Þ k (Mo Ka) (A) T (K) Reflections collected Reflections observed [I > 2rðIÞ] Parameters Ra (observed data) Rw b (observed data) R (all data) Rw (all data) Goodness-of-fit on F 2 Shift/e.s.d. (maximum) 3 ) Largest difference peak and hole (e A P P a R¼ P kFo j jFc k= jFP o j. b wR ¼ f ½wðFo2 Fc2 Þ2 = ½wðFo Þ2 g1=2 ;w ¼ ½r2 ðFo Þ2 þ ðAP Þ2 þ BP 1 , where P ¼ ðFo2 þ 2Fc2 Þ=3; A and B values are 0.0779 and 0.0000.
(5:1 v/v). The source of water could be methanol used for crystallization of the complex. The unit cell parameters and the intensity data were collected on a Bruker SMART APEX CCD diffractometer, equipped with a fine focus 1.75 kW sealed tube Mo Ka X-ray source, with increasing x (width of 0.3°/frame) at a scan speed of 15 s/frame. The SMART software was used for data
acquisition and the SAINT software for data abstraction. Absorptions corrections on the data were made using SADABS [40]. Structure was solved and refined with S H E L X programs [41]. Hydrogen atoms in their calculated positions were refined using a riding model. The non-hydrogen atoms were refined anisotropically. The crystal structure showed the presence of one lattice water molecule in the asymmetric unit. Selected crystallographic data are given in Table 1. Perspective view of the complex was obtained by ORTEP [42].
3. Results and discussion 3.1. Synthetic and general properties The ternary copper(II) complexes 1–4 have been prepared in high yield from a reaction of copper(II) acetate hydrate with the NSO-donor thiosemicarbazone ligand and the NN-donor heterocyclic base. Heterocyclic bases chosen are 1,10-phenanthroline (phen) and its derivatives dipyridoquinoxaline (dpq) and dipyridophenazine (dppz). The presence of extended aromatic ring(s) is expected to enhance the binding propensity with DNA. We have also chosen two thiosemicarbazone ligands having mono-N(4)-substituted methyl or phenyl group to explore their effect on the cleavage activity. The tridentate thiosemicarbazone and didentate heterocyclic base combination gives stable five coordinated structure for the copper(II) center in 1–4. Selected physicochemical data of the complexes are given in Table 2. The one-electron paramagnetic complexes show X-band axial EPR spectra in DMF–toluene glass at 77 K giving gk > g? , indicating a fdx2 y 2 g1 ground state in a square pyramidal geometry. The Ak value for the phen complexes is significantly higher than that for the dpq and dppz complexes. This is indicative of significant distortion from the square pyramidal geometry in 2 and 3 [43,44]. The phen complexes display a
Table 2 Physicochemical data on complexes [Cu(Ln )B] (1–4) kmax / nm ( / M1 cm1 )a
EPRb
d–d band
CT band
gk (104 Ak /cm1 )
g?
1
577 (155)
2.17 (175)
2
720 (105)
3
726 (110)
4
578 (150)
389 (12 400), 318 (17 400), 289 (20 000) 340 (10 250), 397 (3100), 326 (12 100) 386 (3550), 338 (11 450), 322 (14 400) 378 (10 400), 323 (14 600), 309 (15 800)
Complex
a
leff =ðlB Þc
Cu(II)/Cu(I) coupled E1=2 =VðDEp =mVÞ
ipc =ipa
1.98
1.84
0.079 (145)
1.1
2.26 (150)
2.06
1.87
0.080 (140)
1.2
2.26 (150)
2.06
1.82
0.082 (135)
0.9
2.17 (175)
1.98
1.81
0.077 (186)
1.1
In DMF–Tris buffer (pH 7.2, 1:1 v/v). In DMF–toluene glass at 77 K. c At 298 K. d In DMF–0.1 M TBAP. E1=2 ¼ 0:5ðEpa þ Epc Þ, DEp ¼ Epa Epc , where Epa and Epc are anodic and cathodic peak potentials, respectively. Scan rate: 50 mV s1 . ipc and ipa are cathodic and anodic peak currents, respectively. b
A.M. Thomas et al. / Inorganica Chimica Acta 357 (2004) 2315–2323
d–d band in the electronic spectra near 580 nm. A significant shift of this band to lower energy is observed for the dpq and dppz complexes, possibly due to the structural differences among the complexes arising from the extended aromatic rings of these ligands [44]. While the phen complexes show one low energy ligand to copper(II) charge transfer (LMCT) band near 400 nm, the dpq and dppz complexes display two such bands. The lower energy band could be assignable to the sulfur to copper(II) CT transition, while the other may involve the heterocyclic base. The complexes are redox active and show quasireversible cyclic voltammetric response due to the Cu(II)/Cu(I) couple near 0.08 V vs. SCE in DMF–0.1 M TBAP with DEp values ranging between 135 and 186 mV at a scan rate of 50 mV s1 with an ipc =ipa ratio of unity, where ipc and ipa are cathodic and anodic peak currents, respectively. 3.2. Crystal structure Complex [Cu(Ph-tsc)(phen)] H2 O (1 H2 O) has been structurally characterized by single crystal X-ray crystallography. An ORTEP view of the complex is shown in Fig. 1. Selected bond distances and angles are given in Table 3. In the ternary structure, the copper atom is bonded to the thiosemicarbazone ligand showing NSObinding mode at the basal plane in a square pyramidal (4 + 1) geometry in which the NN-donor phen displays axial–equatorial coordination. The copper atom is dis above the N2 SO basal plane. The placed 0.24(1) A trigonality parameter (s) value of 0.32 is indicative of distortion from the square pyramidal geometry [45]. The thiosemicarbazone ligand binds the copper(II) center in a dianionic tridentate fashion with the phenolate oxygen O(1), imine nitrogen N(3) and the sulfur S(1) forming a –N@C(S )-NHPh moiety. The imine bond formation is evidenced from the N(3)–C(19) and N(4)–C(20) dis The dihedral angle tances of 1.283(7) and 1.292(7) A.
Fig. 1. ORTEP view of the complex in 1.H2 O with atom numbering scheme and 50% probability thermal ellipsoids.
2319
Table 3 and angles (°) for [Cu(Ph-tsc)(phen)] Selected bond lengths (A) (1 H2 O) with estimated standard deviations in their parentheses Bond lengths Cu(1)–S(1) Cu(1)–O(1) Cu(1)–N(1) Cu(1)–N(2) Cu(1)–N(3)
Bond angles 2.2489(13) 1.949(3) 2.256(4) 2.042(3) 1.962(3)
S(1)–Cu(1)–O(1) S(1)–Cu(1)–N(1) S(1)–Cu(1)–N(2) S(1)–Cu(1)–N(3) O(1)–Cu(1)–N(1) O(1)–Cu(1)–N(2) O(1)–Cu(1)–N(3) N(1)–Cu(1)–N(2) N(1)–Cu(1)–N(3) N(2)–Cu(1)–N(3)
158.35(11) 103.64(10) 92.46(10) 84.91(10) 97.99(14) 91.24(12) 92.00(13) 77.69(14) 100.79(13) 176.59(15)
between the mean planes of the five and six member chelate rings of the thiosemicarbazone ligand is 15.62°. Near planar arrangement of the chelate rings and the presence of conjugation in the Schiff base could have significant effect on its photosensitizing ability. 3.3. DNA binding and chemical cleavage activity Fluorescence spectral technique has been used to determine the relative binding propensity of the ternary complexes to CT DNA by measuring the emission intensity of ethidium bromide (EB) bound to DNA [46]. Bis(phen)copper(II) complex has been used as a standard. EB does not show any emission in the Tris-buffer medium due to fluorescence quenching by the solvent molecules [47]. However, in the presence of CT DNA, it shows enhanced emission intensity due to its intercalative binding to DNA. Addition of copper complexes results in the competitive binding of the complexes to DNA, thus reducing the emission intensity with either a displacement of the EB from the bound to the free state or the bound copper(II) complex quenching the emission. The decrease in the emission intensity on addition of the complexes is shown in Fig. 2 and the tentative binding order from the slope of the plot is: [Cu(Ph-tsc) (dppz)] (3) > [Cu(phen)2 (H2 O)]2þ > [Cu(Ph-tsc)(dpq)] (2) > [Cu(Ph-tsc)(phen)] (1) > [Cu(Me-tsc)(phen)] (4). Earlier studies on bis-phen copper complexes have shown that this complex binds to DNA either by partial intercalation or binding of one phen ligand to the minor groove, while the other phen making favorable contacts within the groove [3,17,48]. The ancillary ligand plays an important role in DNA groove binding or intercalation. The observed results suggest the greater binding of the dppz complex in comparison to the dpq or phen species. This could be related to its extended aromatic ring or due to its different groove selectivity. We have earlier reported the binding properties of a series of ternary copper(II) complexes having tris(3-phenylpyrazolyl)borate and planar heterocyclic base [44]. It has been observed that while phen and dpq bind at the minor groove, dppz prefers major groove binding. The greater
2320
A.M. Thomas et al. / Inorganica Chimica Acta 357 (2004) 2315–2323
Fig. 2. Effect of addition of [Cu(Ph-tsc)B] [B ¼ phen (1, j); dpq (2, ); dppz (3, N)], [Cu(Me-tsc)(phen)] (4, }) and [Cu(phen)2 (H2 O)](ClO4 )2 (s) to the emission intensity of 125 lM CT DNA-bound ethidium bromide (12.5 lM) in a 5 mM Tris–HCl/50 mM NaCl buffer (pH ¼ 7.2) at 25 °C (*, emission intensity of ethidium bromide in the absence of DNA).
binding of 1 to CT DNA than 4 indicates more favorable contact of Ph-tsc with DNA than its Me-tsc analogue. The oxidative DNA cleavage activity of the complexes was studied by gel electrophoresis using supercoiled (SC) pUC19 DNA in Tris–HCl buffer (pH, 7.2) in the presence of a reducing agent like mercaptopropionic acid (MPA). The complexes show significant cleavage of DNA in dark in the presence of MPA (Table 4, Fig. 3). Control experiments by treating DNA with only MPA or 1 do not show any apparent cleavage of DNA. Comparative studies reveal that the ‘‘chemical nuclease’’ activity of 1–4 is relatively less than that of the bis-(phen)copper(II) species. The observed DNA cleavage order 3 > 2 > 1 > 4 resembles well to their propensity of binding to DNA. Although the true identity of the active species in the oxidative DNA cleavage involving bis(phen)copper and its related complexes is not known, it has been proposed that the Table 4 Cleavage of SC pUC19 DNA (500 ng) by 1–4 (30 lM) in the presence of mercaptopropionic acid (MPA, 50 mM) in the absence of light Serial numbers
1 2 3 4 5 6 7 8
Reaction condition
DNA control DNA + 1 + MPA DNA + 2 + MPA DNA + 3 + MPA DNA + 4 + MPA DNA + bis(phen)Cu(II) + MPA DNA + Distamycin + 2 + MPA DNA + Distamycin + 3 + MPA
% cleavage Form I
Form II
96 59 30 16 70 3
4 41 70 84 30 97
90
10
32
68
oxidative attack at the sugar moiety involves either free hydroxyl radical or a copper bound oxo or hydroxo species [6]. We have carried out mechanistic investigations using DMSO as a scavenger for the hydroxyl radical. The complexes indeed show complete inhibition of cleavage in the presence of DMSO, indicating a reaction pathway that could be similar to the one proposed by Sigman and coworkers [3] for the oxidative cleavage of DNA by bis(phen)copper complex. The groove binding preferences of the complexes are studied using minor groove binder distamycin. While phen and dpq complexes display significant inhibition of cleavage in the presence of distamycin, the dppz complex exhibits only minor inhibition (Table 4). This suggests the minor groove preference of the phen and dpq complexes, while the dppz complex binds at the major groove [44]. 3.4. Photo-induced DNA cleavage The light-induced supercoiled (SC) DNA cleavage activity of the complexes has been studied using UV radiation of wavelength 312 nm and green light of 532 nm (Table 5, Fig. 4). When exposed to UV light for 5 min, the 50 lM solutions of the complexes show 80% SC DNA cleavage for the dpq and dppz complexes, while the phen complex 1 displays moderate cleavage (50%). Among the phen complexes, the Me-tsc species 4 shows significantly less activity than its Ph-tsc analogue. In the ternary structure, the heterocyclic base acts as a DNA binder. The thiosemicarbazone base and the heterocyclic ligands dpq and dppz with their quinoxaline and phenazine moiety, respectively, act as photosensitizer. The observed lower cleavage activity of the Me-tsc complex 4 could be related to its poor binding ability to DNA. The mechanistic aspects of the photo-cleavage reaction have been probed for the major groove binder dppz complex 3 using different reagents. Addition of DMSO, ethanol, mannitol or sodium formate does not inhibit the cleavage activity of the complex, indicating non-involvement of the hydroxyl radical in the photo cleavage reaction. The complex is cleavage inactive under argon atmosphere. Under aerobic conditions, sodium azide shows significant inhibition. The cleavage activity enhances in D2 O. The results suggest the involvement of singlet oxygen (1 O2 ) as a reactive species. Control experiments show that the thiosemicarbazones, phen and dppz, alone are nuclease inactive on photo irradiation at 312 nm. The dpq ligand with its quinoxaline moiety, however, shows significant cleavage at UV radiation. In a recent publication, Toshima and coworkers have shown that quinoxalines, similar to those present in the antitumor antibiotics like triostin and echinomycin, are cleavage active at 365 nm without any external additives by involving C@N bond
A.M. Thomas et al. / Inorganica Chimica Acta 357 (2004) 2315–2323
2321
Fig. 3. Gel diagram showing the oxidative DNA cleavage activity of the complexes 1–4 along with [Cu(phen)2 (H2 O)]2þ in the presence of mercaptopropionic acid (MPA) under different reaction conditions using pUC19 DNA (500 ng) in DMF–Tris–HCl buffer medium (pH 7.2). Lane 1, DNA control; Lane 2, DNA + MPA; Lane 3, DNA + 1; Lane 4, DNA + 1 + MPA; Lane 5, DNA + 2 + MPA; Lane 6, DNA + 3 + MPA; Lane 7, DNA + 4 + MPA; Lane 8, DNA + [Cu(phen)2 (H2 O)]2þ + MPA, Lane 9, DNA + distamycin + 1 + MPA; Lane 10, DNA + distamycin + 2 + MPA; Lane 11, DNA + distamycin + 3 + MPA; Lane 12, DNA + distamycin + 4 + MPA; Lane 13, DNA + distamycin + [Cu(phen)2 (H2 O)]2þ + MPA; Lane 14, DNA + DMSO + 1 + MPA. (complex, 30 lM; MPA, 50 mM; distamycin, 100 lM; DMSO, 4 lL).
Table 5 Photo-cleavage data of SC pUC19 DNA (500 ng) by 1–4 on UV and visible light irradiationa Reaction condition
DNA control DNA + 1 DNA + 2 DNA + 3 DNA + 4 DNA control DNA + dpqb DNA + 1 DNA + 2 DNA + 3 a b
[Complex]/lM
50 50 50 50
200 200 200
k/nm
312 312 312 312 312 532 532 532 532 532
t/min
5 5 5 5 5 30 30 30 30 30
% cleavage Form I
Form II
94 50 25 20 80 90 91 87 49 47
6 50 75 80 20 10 9 13 51 53
t, exposure time. 200 lM.
Fig. 4. (a). Gel diagram showing the photo-cleavage data of SC pUC19 DNA (500 ng) by 1–4 (50 lM for UV and 200 lM for visible radiation) on irradiation with UV light (312 nm, 5 min exposure, Lanes 1–9) and visible light (532 nm, 30 min exposure, Lanes 10–14). Lane 1, DNA control; Lane 2, DNA + Ph-H2 tsc; Lane 3, DNA + dpq; Lane 4, DNA + dppz; Lane 5, DNA + [Cu(phen)2 (H2 O)]2þ ; Lane 6, DNA + 1, Lane 7, DNA + 2; Lane 8, DNA + 3; Lane 9, DNA + 4; Lane 10, DNA control; Lane 11, DNA + dpq; Lane 12, DNA + 1; Lane 13, DNA + 2; Lane 14, DNA + 3 Ligand concentration for control experiments: 50 lM for UV and 200 lM for visible radiation. (b) Gel diagram displaying the cleavage data of SC pUC19 DNA (500 ng) by complex 3 (70 lM) in the presence of various hydroxyl radical scavengers at 312 nm with 5 min exposure time: Lane 1, DNA control; Lane 2, DNA + ethanol (5 lL); Lane 3, DNA + mannitol (100 mM); Lane 4, DNA + sodium formate (100 mM); Lane 5, DNA + DMSO (4 lL); Lane 6, DNA + 3; Lane 7, DNA + ethanol + 3; Lane 8, DNA + mannitol + 3; Lane 9, DNA + sodium formate + 3; Lane 10, DNA + DMSO + 3. (c) Gel diagram displaying the cleavage data of SC pUC19 DNA (500 ng) by complex 3 (90 M) in the presence of various singlet oxygen quenchers at 312 nm with 5 min exposure time: Lane 1, DNA control; Lane 2, DNA + sodium azide (10 mM); Lane 3, DNA + D2 O (14 lL); Lane 4, DNA + 3; Lane 5, DNA + sodium azide + 3; Lane 6, DNA + 3 under argon; Lane 7, DNA + D2 O (14 lL) + 3.
that could generate the photo-excited 3 (n–p ) and/or 3 (p–p ) states causing DNA damage by H-abstraction and/or electron transfer pathways(s) [49]. Control experiments using mono-dpq or mono-dppz copper(II) complex show 90 and 72% cleavage of SC DNA at 312 nm under similar conditions [34]. The reduced activity of the dppz complex than its dpq analogue in
the absence of the thiosemicarbazone ligand could be due to the phenyl ring of the phenazine moiety in dppz reducing the lifetime of its triplet 3 (n–p ) and/or 3 (p–p ) state. It appears from the cleavage data that complexes 2 and 3 are undergoing dual photosensitizing effect, involving the thiosemicarbzone and the heterocyclic ligand. The marginal enhancement of %SC
2322
A.M. Thomas et al. / Inorganica Chimica Acta 357 (2004) 2315–2323
DNA cleavage observed for the dppz complex than its dpq analogue may be attributed to the effect of their different groove selectivity. The cleavage data also suggest that the conjugation present in the thiosemicarbazone ligand makes this ligand inefficient possibly by reducing the triplet state lifetime of the sulfur ligand in an enolic form. As a consequence, the photosensitizing effect is more prominent for the quinoxaline and phenazine moieties than that of the sulfur ligand. We have recently reported the photonuclease activity of mono-(phen)copper(II) complex containing NSO-donor Schiff base, 2-(methylthio)ethylsalicylaldimine [22]. Absence of any conjugation between the thio group and the imine moiety enhances the photosensitizing effect. We have probed the photo-nuclease activity of the dpq and dppz complexes using a visible light source of 532 nm. Moderate cleavage is observed for 2 and 3 on irradiation at this wavelength (Table 5, Fig. 4). Control experiments show that the ligands alone are cleavage inactive at this wavelength. The results indicate the possible involvement of the copper(II) center in the formation of singlet oxygen, involving initial photosensitization process of the sulfur ligand as well as the heterocyclic base. The moderate cleavage activity could also be due to the possibility of the sulfur atom of the thiosemicarbazone ligand undergoing oxidation in the presence of singlet oxygen [50]. Mechanistic studies indicate the formation of singlet oxygen as the active species cleaving DNA as a major pathway. The alternate pathway leading to the formation of hydroxyl or copper-oxo species, by involving the photo-activated ligand reducing the copper from +2 to +1 oxidation state in a process similar to that proposed by Sigman and coworkers for the ‘‘chemical nuclease’’ reaction of the bis-phen copper complex, seems to be less probable [3,17].
4. Conclusions New ternary copper(II) complexes having thiosemicarbazones as photosensitizer and phenanthroline bases as DNA binder are prepared, structurally characterized, and their DNA cleavage properties studied. The dpq and dppz complexes show significant cleavage of SC DNA on irradiation with green light in the absence of any additives. The dpq and dppz complexes also show dual photosensitizing effect by involving the thiosemicarbazone ligand and the quinoxaline or phenazine moiety of the heterocyclic base. The dpq and phen complexes are minor groove binder, while the dppz complex binds at the major groove of DNA. Mechanistic studies reveal possible involvement of hydroxyl radical and/or copper bound oxo/hydroxo species in the oxidative cleavage of DNA in the presence of merca-
ptopropionic acid. The photocleavage reactions under UV or visible light irradiation involve singlet oxygen as the DNA cleaving agent. The present results are of significance, considering the pharmacological and antitumor activity of thiosemicarbazones and the antitumor activities of quinoxaline antibiotics. Our observation of the copper(II) center assisting the photo-cleavage of DNA is of significance as the same metal center, in contrast, is known to reduce the cleavage activity of porphyrin-copper(II) complexes by significantly reducing the 3 (p–p ) state lifetime of the porphyrin bases [23]. We have observed a significant effect of conjugation in the sulfur containing Schiff base in reducing the cleavage efficiency. The results are of importance in designing copper-based complexes containing sulfur containing ligands for PDT applications.
5. Supplementary material Crystallographic data (excluding structure factors) for the structure have been deposited with the Cambridge Crystallographic Data Center, CCDC reference no. 215179. Copies of this information may be obtained free of charge from The Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: (internat.) +441223-336-033; e-mail: [email protected] or http:// www.ccdc.cam.ac.uk).
Acknowledgements We thank the Council of Scientific and Industrial Research (CSIR), New Delhi, and the Department of Science and Technology (DST), Government of India, for the financial support; DST for the CCD diffractometer facility; the Alexander von Humboldt Foundation, Germany, for donation of an electroanalytical system; and the Convener, Bioinformatics Center, Indian Institute of Science, Bangalore, for database search.
References [1] (a) B. Meunier, Chem. Rev. 92 (1992) 1411; (b) G. Pratviel, J. Bernadou, B. Meunier, Angew. Chem., Int. Ed, Engl. 34 (1995) 746; (c) M. Pitie, J.D.V. Horn, D. Brion, C.J. Burrows, B. Meunier, Bioconjugate Chem. (2000) 892. [2] K.E. Erkkila, D.T. Odom, J.K. Barton, Chem. Rev. 99 (1999) 2777. [3] (a) D.S. Sigman, T.W. Bruce, A. Mazumder, C.L. Sutton, Acc. Chem. Res. 26 (1993) 98; (b) D.S. Sigman, A. Mazumder, D.M. Perrin, Chem. Rev. 93 (1993) 2295. [4] S.J. Lippard, Chem. Rev. 99 (1999) 2467.
A.M. Thomas et al. / Inorganica Chimica Acta 357 (2004) 2315–2323 [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
[15]
[16]
[17]
[18] [19]
[20]
[21] [22]
[23]
[24]
J. Reedijk, J. Inorg. Biochem. 86 (2001) 89. C.J. Burrows, J.G. Muller, Chem. Rev. 98 (1998) 1109. B. Armitage, Chem Rev. 98 (1998) 1171. W.K. Pogozelski, T.D. Tullius, Chem. Rev. 98 (1998) 1089. D.R. Mc Millin, K.M. Mc Nett, Chem. Rev. 98 (1998) 1201. S.J. Lippard, J.M. Berg, Prinicples of Bioinorganic Chemistry, University Science Books, CA, 1994. R. Ackroyd, C. Kelty, N. Brown, M. Reed, Photochem. Photobiol. 74 (2001) 656. B.W. Henderson, T.J. Dougherty, Photochem. Photobiol. 55 (1992) 145. M.C. De Rose, R.J. Crutchley, Coord. Chem. Rev. 233–234 (2002) 351. (a) E.D. Sternberg, D. Dolphin, C. Br€ uckner, Tetrahedron 54 (1998) 4151; (b) J.L. Sessler, G. Hemmi, T.D. Mody, T. Murai, A. Burrell, S.W. Young, Acc. Chem. Res. 27 (1994) 43; (c) H. Ali, J.E. Van Lier, Chem. Rev. 99 (1999) 2379. B.W. Henderson, T.M. Busch, L.A. Vaughan, N.P. Frawley, D. Babich, T.A. Sosa, J.D. Zollo, A.S. Dee, M.T. Cooper, D.A. Bellnier, W.R. Greco, A.R. Oseroff, Cancer Res. 60 (2000) 525. (a) O. Baudoin, M.-P. Teulade-Fichou, J.-P. Vigneron, J.-M. Lehn, Chem. Commun. (1998) 2349; (b) M. Gonzalez-Alvarez, G. Alzuet, J. Borras, M. Pitie, B. Meunier, J. Biol. Inorg. Chem. 8 (2003) 644; (c) J.H Kim, S.H. Kim, Chem. Lett. 32 (2003) 490; (d) A. Garca-Raso, J.J. Fiol, B. Adrover, V. Moreno, I. Mata, E. Espinosa, E. Molins, J. Inorg. Biochem. 95 (2003) 77; (e) L.-P. Lu, M.-L. Zhu, P. Yang, J. Inorg. Biochem. 95 (2003) 31; (f) J.A. Cowan, Chem. Commun. (2001) 1490. (a) D.S. Sigman, Biochemistry 29 (1990) 9097; (b) T.B. Thederahn, M.D. Kuwabara, T.A. Larsen, D.S. Sigman, J. Am. Chem. Soc. 111 (1989) 4941; (c) O. Zelenko, J. Gallagher, D.S. Sigman, Angew. Chem., Int. Ed. Engl. 36 (1997) 2776; (d) M.M. Meijler, O. Zelenko, D.S. Sigman, J. Am. Chem. Soc. 119 (1997) 1135. E.L. Hegg, J.N. Burstyn, Coord. Chem. Rev. 173 (1998) 133. (a) D.K. Chand, H.-J. Schneider, A. Bencini, A. Bianchi, C. Giorgi, S. Ciattini, B. Valtancoli, Chem. Eur. J. 6 (2000) 4001; (b) R. Hettich, H.-J. Schneider, J. Am. Chem. Soc. 119 (1997) 5638. (a) J.A. Cowan, Chem. Rev. 98 (1998) 1067; (b) F.H. Westheimer, Science 235 (1987) 1173; (c) D.E. Wilcox, Chem. Rev. 96 (1996) 2435; (d) M. Komiyama, J. Sumaoka, Curr. Opin. Chem. Biol. 2 (1998) 751. H.J. Eppley, S.M. Lato, A.D. Ellington, J.M. Zaleski, Chem. Commun. (1999) 2405. (a) S. Dhar, A.R. Chakravarty, Inorg. Chem. 42 (2003) 2483; (b) A.K. Patra, S. Dhar, A.R. Chakravarty, Chem. Commun. (2003) 1562; (c) S. Dhar, D. Senapati, P.K. Das, P. Chattopadhyay, M. Nethaji, A.R. Chakravarty, J. Am. Chem. Soc. 125 (2003) 12118; (d) S. Dhar, D. Senapati, P.A.N. Reddy, P.K. Das, A.R. Chakravarty, Chem. Commun. (2003) 2452. (a) D. Praseuth, A. Gaudemer, J.-B. Verlhac, I. Kraljic, I. Sissoeff, E. Guille, Photochem. Photobiol. 44 (1986) 717; (b) S. Somer, C. Rimington, J. Mohan, FEBS Lett. 172 (1984) 267. M. Baldini, M.B. Ferrari, F. Bisceglie, G. Pelosi, S. Pinelli, P. Tarasconi, Inorg. Chem. 42 (2003) 2049.
2323
[25] Q. Xuhong, H. Tian-Bao, W. Dong-zhi, Z. Dong-Hui, F. Mingcai, Y. Wei, J. Chem. Soc., Perkin Trans. 2 (2000) 715. [26] A. Jakobs, J. Piette, J. Photochem. Photobiol. B. Biol (1994) 2219. [27] A. Jakobs, J. Piette, J. Med. Chem. 38 (1995) 869. [28] (a) S. Padhye, S.B. Kauffman, Coord. Chem. Rev. 63 (1985) 127; (b) D.X. West, A.E. Liberta, S.B. Padhye, R.C. Chikate, P.B. Sonawane, A.S. Kumbhar, R.G. Yerande, Coord. Chem. Rev. 123 (1993) 49. [29] R.W. Byrnes, M. Mohan, W.E. Antholine, R.X. Xu, D.H. Petering, Biochemistry 29 (1990) 7046. [30] (a) Z. Afrasiabi, E. Sinn, S. Padhye, S. Dutta, S. Padhye, C. Newton, C.E. Anson, A.K. Powell, J. Inorg. Biochem. 95 (2003) 306; (b) A.I. Matesanz, J.M. Perez, P. Navarro, J.M. Moreno, E. Colacio, P. Souza, J. Inorg. Biochem. 76 (1999) 29. [31] D.K. Demertzi, P.N. Yadav, M.A. Demertzis, M. Coluccia, J. Inorg, Biochem. 78 (2000) 347. [32] Z. Lakovidou, A. Papgeorgiou, M.A. Demertzis, E. Mioglou, D. Mourelatos, A. Kotsis, P.N. Yadav, D.K. Demertzi, Anti Cancer Drugs 12 (2001) 65. [33] (a) K.H. Reddy, P.S. Reddy, P.R. Babu, Trans. Met. Chem. 25 (2000) 154; (b) K.H. Reddy, P.S. Reddy, P.R. Babu, J. Inorg. Biochem. 77 (1999) 169. [34] A.M. Thomas, M. Nethaji, S. Mahadevan, A.R. Chakravarty, J. Inorg. Biochem. 94 (2003) 171. [35] (a) H. Nakai, Bull. Chem. Soc. Jpn. 44 (1971) 2412; (b) J. Foley, D. Kennefick, D. Phelan, S. Tyagi, B. Hathaway, J. Chem. Soc. Dalton Trans. (1983) 2333. [36] D.L. Klayman, J.F. Brtosevich, T.S. Griffin, C.J. Mason, J.P. Scovill, J. Med. Chem. 22 (1997) 855. [37] J. Bernadou, G. Pratviel, F. Bennis, M. Girardet, B. Meunier, Biochemistry 28 (1989) 7268. [38] (a) E. Amouyal, A. Homsi, J.-C. Chambron, J.-P. Sauvage, J. Chem. Soc., Dalton Trans. (1990) 1841; (b) J.E. Dickenson, L.A. Summers, Aust. J. Chem. 23 (1970) 1023. [39] M.E. Reichmann, S.A. Rice, C.A. Thomas, P. Doty, J. Am. Chem. Soc. 76 (1954) 3047. [40] R. Blessing, Acta Crystallogr. Sect A. 51 (1995) 33. [41] G.M. Sheldrick, S H E L X -97, Programs for Crystal Structure Solution and Refinement, University of G€ ottingen, Germany, 1997. [42] C.K. Johnson, ORTEP, Report ORNL – 5138, Oak Ridge National Laboratory, Oak Ridge, TN, 1976. [43] A.D. Naik, P.A.N. Reddy, M. Nethaji, A.R. Chakravarty, Inorg. Chim. Acta 349 (2003) 149. [44] S. Dhar, P.A.N. Reddy, M. Nethaji, S. Mahadevan, M.K. Saha, A.R. Chakravarty, Inorg. Chem. 41 (2002) 3469. [45] A.W. Addison, T.N. Rao, J. Reedijk, J.V. Riju, G.C. Verschoor, J. Chem. Soc., Dalton Trans. (1984) 1349. [46] L.C. Zheng, W. Jigui, W. Liufang, R. Min, J. Naiyong, G. Jie, J. Inorg. Biochem. 73 (1999) 195. [47] M.J. Waring, J. Mol. Biol. 13 (1965) 269. [48] (a) B.C. Bales, M. Pitie, B. Meunier, M.M. Greenberg, J. Am. Chem. Soc. 124 (2002) 9062; (b) J.M. Veal, K. Merchant, R.L. Rill, Nucleic Acids Res. 19 (1991) 3383. [49] K. Toshima, R. Takano, T. Ozawa, S. Matsumura, Chem. Commun. (2002) 212. [50] F. Champloy, N. Benali-Cherif, P. Bruno, I. Blain, M. Pierrot, M. Reglier, Inorg. Chem. 37 (1998) 3910.