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DNA-binding and photocleavage studies of cobalt(III) polypyridyl complexes: [Co(phen)2IP]3+ and [Co(phen)2PIP]3+
Journal of Inorganic Biochemistry 83 (2001) 49–55 www.elsevier.nl / locate / jinorgbio
DNA-binding and photocleavage studies of cobalt(III) polypyridyl complexes: [Co(phen) 2 IP] 31 and [Co(phen) 2 PIP] 31 Qian-Ling Zhang, Jin-Gang Liu, Hui Chao, Gen-Qiang Xue, Liang-Nian Ji* Department of Chemistry, Zhongshan University, Guangzhou 510275, PR China Received 29 March 2000; received in revised form 9 June 2000; accepted 22 June 2000
Abstract Two complexes of [Co(phen) 2 IP] 31 (IP5imidazo[4,5-f ][1,10]phenanthroline) and [Co(phen) 2 PIP] 31 (PIP52-phenylimidazo[4,5f ][1,10]phenanthroline) have been synthesized and characterized by UV/ VIS, IR, EA and mass spectra. The binding of the two complexes with calf thymus DNA has been investigated by absorption spectroscopy, cyclic voltammetry, viscosity measurements and DNA cleavage assay. The spectroscopic studies together with cyclic voltammetry and viscosity experiments support that both of the complexes bind to CT DNA by intercalation via IP or PIP into the base pairs of DNA. [Co(phen) 2 PIP] 31 binds more avidly to CT DNA than [Co(phen) 2 IP] 31 , which is consistent with the extended planar and p system of PIP. Noticeably, the two complexes have been found to be efficient photosensitisers for strand scissions in plasmid DNA. 2001 Elsevier Science B.V. All rights reserved. Keywords: Cobalt(III) complexes; DNA-binding; Photocleavage
1. Introduction The interaction of transition metal complexes with DNA has been extensively studied in the past few years. Metal complexes of the type [M(LL) 3 ] n1 where LL is either 1,10-phenanthroline (phen) or a modified phen ligand, are particularly attractive species to recognize and cleave DNA [1–4]. In the early 1980s, Barton et al. demonstrated that tris(phenanthroline) complexes of ruthenium(II) display enantiomeric selectivity in binding to DNA, which can be served as spectroscopic probes in solution to distinguish right- and left-handed DNA helices [5]. Then they found that tris(phenanthroline) complexes of cobalt(III) can cleave DNA when irradiated at 254 nm. Furthermore, they conducted the cleavage reactions by using the high stereospecificity of the tris(diphenylphenanthroline) (DIP) metal isomers. The cleavage reaction is also stereospecific. Incubation of pCo1E1 DNA with L-Co(DIP) 31 yielded no 3 appreciable reaction, whereas incubation with DCo(DIP) 31 showed efficient nicking activity. These find3 ings underscore the importance of an intimate association of the metal with the duplex. The high level of recognition of DNA conformation by these chiral inorganic complexes *Corresponding author. Tel.: 186-20-8411-0115; fax: 186-20-84035497. E-mail address: [email protected] (L.-N. Ji).
suggested the powerful application of stereospecificity in DNA drug design [6]. The features common to these complexes are that the molecule has a high affinity for double-stranded DNA and that the molecule also binds a redox-active metal ion cofactor. The ligands or the metal in these complexes can be varied in an easily controlled manner to facilitate an individual application. All the studies reveal that modification of the metal or ligands would lead to subtle or substantial changes in the binding modes, location and affinity [7,8], giving changes to explore various valuable conformation or site-specific DNA probes and potential chemotherapeutical agents. Currently, much attention has been paid to the complexes of Ru(II) [9–15], but the complexes metal ion other than ruthenium(II) have attracted much less attention. We chose to concentrate our work on complexes of cobalt(III), which have the same interesting characteristics and DNA cleaving properties, but have not received as much attention as the Ru(II) systems [6,16–19]. Clearly further studies using various metals to evaluate the effect of intercalated ligand on the DNA binding and cleavage mechanisms are necessary. In this paper, we report the synthesis, characterization of the two complexes [Co(phen) 2 IP] 31 (IP5imidazo[4,5f ][1,10]phenanthroline) and [Co(phen) 2 PIP] 31 (PIP52phenylimidazo[4,5-f ][1,10]phenanthroline), in which PIP possesses a greater planar area and extended p system than that of IP, and their DNA-binding properties are revealed
0162-0134 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0162-0134( 00 )00132-X
Q.-L. Zhang et al. / Journal of Inorganic Biochemistry 83 (2001) 49 – 55
50
by cyclic voltammetry, electronic absorption, luminescent spectra and viscosity measurements. The photochemical DNA cleavage of the two complexes is also demonstrated. These studies are necessary for the further comprehension of binding of transition metal complexes to DNA, they also can be served as complementary studies for the corresponding complexes of ruthenium.
2. Experimental
2.1. Materials and methods All materials were purchased and used without further purification unless otherwise noted. IP and PIP [20], cis[Co(phen) 2 Cl 2 ]Cl?3H 2 O [21] and [Co(phen) 3 ]Cl 3 [22] were prepared by the methods described previously. The structures of ligands are shown in Fig. 1. All the experiments involving the interaction of the complexes with DNA were carried out in doubly distilled buffer (5 mM Tris–HCl, 50 mM NaCl, pH 7.2). A solution of calf thymus DNA in the buffer gave a ratio of UV absorbance at 260 and 280 nm of about 1.8–1.9:1, indicating that the DNA was sufficiently free of protein [23]. The DNA
concentration per nucleotide was determined by absorption spectroscopy using the molar absorption coefficient (6600 M 21 cm 21 ) at 260 nm [24].
2.1.1. Synthesis of [ Co( phen)2 IP]( ClO4 )3?2 H2 O A mixture of [Co(phen) 2 Cl 2 ]Cl (0.578 g, 1.0 mmol) and IP (0.33 g, 1.5 mmol) in ethanol (50 ml) was refluxed for about 3 h. After filtration, the complex was precipitated upon addition of a saturated ethanolic solution of NaClO 4 . The complex was filtered and further dried under vacuum before recrystallization (acetone–ether). Yield: 0.432 g, 44%. (Found: C, 45.45; H, 3.06; N, 11.83. Calc. for C 37 N 8 H 28 Cl 3 O 14 Co: C, 45.61; H, 2.88; N, 11.51%). lmax / 21 21 nm (´ / M cm ) (water). 272 (44 400), 260(sh), 220 (69 650). nmax / cm 21 , 3446 w(br), 3058 w, 1609 m, 1468 m, 1371 m, 1090 vs, 810 m, 782 s, 706 m and 630 s. FAB-MS: m /z5838 (M-ClO 4 ), 738 (M-2ClO 4 ) and 639 (M-3ClO 4 ). 2.1.2. Synthesis of [ Co( phen)2 PIP]( ClO4 )3?5 H2 O [Co(phen) 2 PIP](ClO 4 ) 3 ?5H 2 O was prepared in a similar way to that of [Co(phen) 2 IP](ClO 4 ) 3 ?2H 2 O. Yield: 0.583 g, 52.8%. (Found: C, 46.49; H, 3.59; N, 10.50. Calc. for C 43 N 8 H 38 Cl 3 O 17 Co: C, 46.76; H, 3.44; N, 10.15%). lmax / 21 21 nm (´ / M cm ) (water). 280 (44 700), 218 (64 850). 21 nmax / cm , 3416 w(br), 3057 w, 1609 m, 1468 m, 1370 m, 1082 vs, 807 m, 779 s, 704 m and 632 s. FAB-MS: m /z5915 (M-ClO 4 ), 814 (M-2ClO 4 ) and 715 (M-3ClO 4 ). Caution: Perchlorate salts of metal complexes with organic ligands are potentially explosive. Only small amounts of the material should be prepared and handled with great care. 2.2. Physical measurements
Fig. 1. Chemical structures of the ligands.
Elemental analyses (C, H and N) were performed on a Perkin-Elmer 240 Q elemental analyser. Infrared spectra were obtained on a Nicolet 170X-FTIR spectrometer as KBr discs. UV-VIS spectra were recorded on a Shimadzu MPS-2000 spectrophotometer. Fast atomic bombardment mass spectra (FAB-MS) were obtained on a VG ZAB-HS spectrometer in a 3-nitrobenzyl alcohol matrix. Cyclic voltammetry was performed on an EGandG PAR 273 polarographic analyser and 270 universal programmer. The supporting electrolyte was 50 mM NaCl, 10 mM Tris, pH 7.2. All samples were purged with nitrogen prior to measurements. A standard three-electrode system was used comprising an Au working electrode, platinum-wire auxiliary electrode and a saturated calomel reference electrode (SCE). For the absorption spectra, equal solution of DNA was added to both complex solution and reference solution to eliminate the absorbance of DNA itself. Viscosity experiments were carried on an Ubbelohde viscometer, immersed in a thermostatted water-bath main-
Q.-L. Zhang et al. / Journal of Inorganic Biochemistry 83 (2001) 49 – 55
tained at 3060.18C. DNA samples approximately 200 base pairs in average length were prepared by sonication in order to mimimize complexities arising from DNA flexibility [25]. Data were presented as (h /h0 )1 / 3 versus the concentration of Co(III) complexes, where h is the viscosity of DNA in the presence of complex, and h0 is the viscosity of DNA alone. Viscosity values were calculated from the observed flow time of DNA-containing solutions (t.100 s) corrected for the flow time of buffer alone (t 0 ), h 5 t 2 t 0 [26]. For the gel electrophoresis experiments, supercoiled pBR 322 DNA (100 mM) was treated with Co(III) complexes in 50 mM Tris–HCl, 18 mM NaCl buffer, pH 7.2, and the solutions were then irradiated at room temperature with a UV lamp (302 nm, 10 W). The samples were analysed by electrophoresis for 2.5 h at 40 V on a 0.8% agarose gel in Tris–acetic acid–EDTA buffer, pH 7.2. The gel was stained with 1 mg / ml ethidium bromide and photographed under UV light.
3. Results and discussion
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Epa , is 0.095 V, in the absence of DNA. The presence of DNA in the solution at the same concentration of [Co(phen) 2 IP] 31 causes a considerable decrease in the voltammetric current coupled with a slight shift in the E1 / 2 (E1 / 2 50.077 V) to less negative potential (Fig. 2B). For the CV behavior of [Co(phen) 2 PIP] 31 under similar conditions, in the absence of DNA, Epc 5 20.05 V and Epa 50.08 V. The formal potential E1 / 2 is 0.015 V. Upon addition of CT DNA, both the anodic and cathodic peak potential shift to some more negative values (E1 / 2 5 2 0.032 V) coupled with the decrease of its voltammetric current, which is similar to that of [Co(phen) 2 IP] 31 observed, except that the magnitude of the voltammetric current decrease is some more appreciable than that of the [Co(phen) 2 IP] 31 . The drop of the voltammetric currents in the presence of CT DNA can be attributed to diffusion of the metal complex bound to the large, slowly diffusing DNA molecule. The more pronounced decrease of the peak currents upon addition of CT DNA, which are observed for [Co(phen) 2 PIP] 31 over that of [Co(phen) 2 IP] 31 , may indicate the binding affinity of the former to DNA is higher than that of the latter.
3.1. Cyclic voltammetry Typical cyclic voltammetric (CV) behavior of 0.1 mM [Co(phen) 2 IP] 31 in the absence and presence of CT DNA is shown in Fig. 2. The cyclic voltammogram of [Co(phen) 2 IP] 31 in the absence of DNA (Fig. 2A) featured reduction of 31 to the 21 form at a cathodic peak potential, Epc of 0.04 V versus SCE. Reoxidation of 21 occurred, upon scan reversal, at 0.15 V. The separation of the anodic and cathodic peak potentials, DEp 5110 mV, indicated a irreversible redox process. The formal potential E 0 9 (or voltammetric E1 / 2 ), taken as the average of Epc and
Fig. 2. Cyclic voltammograms of [Co(phen) 2 IP] 31 in the absence (A) and presence (B) of DNA in 50 mM NaCl, 10 mM Tris, pH 7.2. [Co]5100 mM, [DNA] / [Co]520. Scan rates, 200 mV s 21 .
3.2. Electronic absorption spectra The application of electronic absorption spectroscopy in DNA-binding studies is one of the most useful techniques [27–29]. Complex binding with DNA through intercalation usually results in hypochromism and bathchromism, due to the intercalative mode involving a strong stacking interaction between an aromatic chromophore and the base pairs of DNA. The extent of the hypochromism commonly parallels the intercalative binding strength. The absorption spectra of the complexes in the absence and presence of calf thymus DNA are illustrated in Fig. 3. The electronic absorption spectra of the two complexes in Tris buffer are similar in shape to that of [Co(phen) 3 ] 31 . In the UV region, the intense absorption bands observed in the Co(III) complexes are attributed to intraligand p2p* transition of the coordinated groups. Addition of increasing amounts of CT DNA results in hypochromism and moderate bathochromic shift in the UV spectra of both [Co(phen) 2 IP] 31 and [Co(phen) 2 PIP] 31 . According to the data presented in Table 1, it seems that the spectral perturbation of the three complexes upon addition of DNA follows: [Co(phen) 2 PIP] 31 . [Co(phen) 2 IP] 31 . [Co(phen) 3 ] 31 . These spectral characteristics may suggest a mode of binding that involves a stacking interaction between the complex and the base pairs of DNA. In order to quantitatively compare the binding strength of the two complexes, the intrinsic binding constants K of the two complexes with CT-DNA were determined according to the following equation [30] through a plot of [DNA] /(´a 2 ´f ) versus [DNA].
Q.-L. Zhang et al. / Journal of Inorganic Biochemistry 83 (2001) 49 – 55
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Fig. 3. Absorption spectra of [Co(phen) 2 IP] 31 (A) and [Co(phen) 2 PIP] 31 (B) in the absence (———) and presence (- - -) of DNA with subtraction of the DNA absorbance. [Co]520 mM, [DNA] / [Co]55, 10, 15. Arrow shows the absorbance changes upon increasing DNA concentrations. Insets: plots of [DNA] /(´a 2 ´f ) versus [DNA] for the titration of DNA with complexes; (d) experimental data points; solid line, linear fitting of the data.
[DNA] /(´a 2 ´f ) 5 [DNA] /(´b 2 ´f ) 1 1 /(K(´b 2 ´f )) Where [DNA] is the concentration of DNA in base pairs, the apparent absorption coefficient ´a , ´f and ´b correspond to A obsd / [Co], the extinction coefficient for the free cobalt complex and the extinction coefficient for the cobalt complex in the fully bound form, respectively. In plots [DNA] /(´a 2 ´f ) versus [DNA], K is given by the ratio of slope to intercept. Intrinsic binding constants K of [Co(phen) 2 IP] 31 and [Co(phen) 2 PIP] 31 were obtained about 1.3360.1310 5 and 2.1560.2310 5 M 21 from the decay of the absorbance, respectively, and the binding sites are 4 and 6, respectively. The binding constants indicate that [Co(phen) 2 PIP] 31 binds more strongly than [Co(phen) 2 IP] 31 . This result is expected, since PIP posses-
Table 1 Absorption spectroscopic properties of the Co(III) complexes on binding to DNA Complex
[Co(phen) 2 IP] 31 [Co(phen) 2 PIP] 31 [Co(phen) 3 ] 31 b a b
Absorption lmax (nm) Free
Bound a
220 272 218 280 219 274
223 274 223 282 223 276
[Co]520 mM at [DNA] / [Co]515. From Ref. [19].
Dl
Hypochromicity (%)
3 2 5 2 4 2
19.9 27.9 41.6 28.3 9.6 15.2
ses a greater planar area and extended p system than that of IP, which will lead to PIP penetrating more deeply into, and stacking more strongly with the base pairs of DNA.
3.3. Fluorescence spectroscopic studies The complexes [Co(phen) 2 IP] 31 and [Co(phen) 2 PIP] 31 can emit luminescence in Tris buffer at ambient temperature with maxima at 409 and 410 nm, respectively. Binding of both complexes to DNA was found to increase the fluorescence intensity. The emission spectra of both complexes in the absence and presence of CT DNA are shown in Fig. 4. The plots of the relative intensity versus the ratio of [DNA] / [Co] are also inserted in Fig. 4. Upon addition of CT DNA, the emission intensity increases steadily and reaches 1.4 times larger than that of in the absence of DNA for [Co(phen) 2 IP] 31 , and 2.0 times for [Co(phen) 2 PIP] 31 , respectively, at the ratio of [DNA] / [Co]520. The extent of enhancement increases on going from [Co(phen) 2 IP] 31 to [Co(phen) 2 PIP] 31 , which is consistent with the above absorption spectra results.
3.4. Viscosity studies Further clarification of the interaction between the two complexes and DNA was carried out by viscosity measurements. Optical photophysical probes are necessary, but not sufficient clues to support a binding model. Hydrodynamic measurements that sensitive to length change (i.e., viscosity and sedimentation) are regarded as the least ambiguous
Q.-L. Zhang et al. / Journal of Inorganic Biochemistry 83 (2001) 49 – 55
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ligand could bind (or kink) the DNA helix, reduce its effective length and, concomitantly, its viscosity [26,31]. The effects of both complexes and [Co(phen) 3 ] 31 on the viscosity of rod-like DNA are shown in Fig. 5. For [Co(phen) 2 IP] 31 and [Co(phen) 2 PIP] 31 , the viscosity of DNA increases with the increase of the concentration of the complexes, which is similar to that of proven DNA intercalator [Ru(phen) 2 DPPZ] 21 [32]. Both complexes increase the relative viscosity of DNA in a manner consistent with binding by classical intercalation. This result also parallels the pronounced hypochromism and spectral red shift and emission enhancement of both complexes, whereas [Co(phen) 3 ] 31 does not extend DNA helix length. On the basis of the viscosity results, it seems that [Co(phen) 3 ] 31 binds with DNA through groove binding. Although Barton and co-workers have proposed that [Co(phen) 3 ] 31 intercalated into double-stranded DNA [33], this viscosity experiment shows that [Co(phen) 3 ] 31 is not a DNA intercalation agent.
3.5. Photoactivated cleavage of pBR 322 DNA by Co( III) complexes There has been considerable interest in DNA endonucleolytic cleavage reactions that are activated by metal ions [34,35]. The delivery of high concentrations of metal ion to the helix, in locally generating oxygen or hydroxide radicals, yields an efficient DNA cleavage reaction. DNA cleavage was monitored by relation of supercoiled circular pBR 322 (Form I) into nicked circular (Form II) and linear (Form III). When circular plasmid DNA is subjected to electrophoresis, relatively fast migration will be observed
Fig. 4. Emission spectra of [Co(phen) 2 IP] 31 (A) and [Co(phen) 2 PIP] 31 (B) in aqueous buffer (Tris 5 mM, NaCl 50 mM, pH 7.2) at 298 K in the presence of CT DNA. [Co]520 mM, [DNA] / [Co]50, 5, 10, 15, 20. lex 5320 nm. Arrow shows the intensity changes upon increasing concentration. Inset: plots of relative integrated emission intensity versus [DNA] / [Co].
and the most critical tests of binding in solution in the absence of crystallographic structural data [26,31]. A classical intercalation model results in lengthening the DNA helix, as base pairs are separated to accommodate the binding ligand, leading to the increase of DNA viscosity. In contrast, a partial and / or non-classical intercalation of
Fig. 5. Effect of increasing amounts of [Co(phen) 2 IP] 31 (j), [Co(phen) 2 PIP] 31 (d) and [Co(phen) 3 ] 31 (m) on the relative viscosities of CT DNA at 3060.18C.
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Q.-L. Zhang et al. / Journal of Inorganic Biochemistry 83 (2001) 49 – 55
for the supercoiled form (Form I). If scission occurs on one strand (nicking), the supercoils will relax to generate a slower-moving open circular form (Form II) [6]. If both strands are cleaved, a linear form (Form III) will be generated that migrates between Forms I and II. Fig. 6 shows the gel electrophoretic separations of plasmid pBR
322 DNA after incubation with Co(III) complexes and irradiation at 302 nm. Fig. 6A,B reveals the conversion of Form I to II after 60 min irradiation in the presence of varying concentrations of [Co(phen) 2 IP] 31 and 31 [Co(phen) 2 PIP] . It can be seen that with increasing the concentration of [Co(phen) 2 IP] 31 and [Co(phen) 2 PIP] 31 , Form II increases gradually, while Form I diminishes gradually. With increasing irradiation time, Form I of pBR 322 DNA diminishes gradually, whereas the amount of Form II increases (Fig. 6C). This is the result of singlestranded cleavage of pBR 322 DNA. It can also be seen in Fig. 6C that neither irradiation of DNA at 302 nm without Co(III) nor incubation with Co(III) without light yields significant strand scission. It is likely that the reduction of Co(III) is the important step leading to DNA cleavage. Further study is necessary to clarify the reaction mechanism.
4. Conclusions Two complexes [Co(phen) 2 IP] 31 and [Co(phen) 2 PIP] 31 are synthesized and characterized. Spectroscopic studies together with cyclic voltammetry and viscosity experiments support that both of the complexes bind to CT DNA by intercalation via IP or PIP into the base pairs of DNA. The intrinsic binding constants indicate that [Co(phen) 2 PIP] 31 binds more avidly to CT DNA than [Co(phen) 2 IP] 31 , which is consistent with the extended planar and p system of PIP. Noticeably, both complexes have been found to promote cleavage of plasmid pBR 322 DNA from the supercoiled form I to the open circular form II upon irradiation, which may be taken as the potential DNA cleavage reagent.
5. Abbreviations CT DNA IL IP phen PIP SCE Fig. 6. (A) Photoactivated cleavage of pBR 322 DNA in the presence of [Co(phen) 2 IP] 31 and light after 60 min irradiation at 302 nm. DNA alone (lane 0), the concentration of [Co(phen) 2 IP] 31 was 10, 20, 30, 40, 50 and 60 mM (lanes 1–6, left to right). (B) Photoactivated cleavage of pBR 322 DNA in the presence of [Co(phen) 2 PIP] 31 and light after 60 min irradiation at 302 nm. The concentration of [Co(phen) 2 PIP] 31 was 10, 20, 40, 50 and 60 mM (lanes 1–5, left to right). (C) Photoactivated cleavage of pBR 322 in the presence of [Co(phen) 2 IP] 31 (20 mM) after irradiation at 302 nm for 0, 20, 40, 60, 80, 100 and 120 min (lanes 1–7, left to right), DNA alone (lane 0).
Calf thymus DNA Intraligand Imidazo[4,5-f ][1,10]phenanthroline 1,10-Phenanthroline 2-Phenylimidazo[4,5-f ][1,10]phenanthroline Saturated calomel electrode
Acknowledgements We are grateful to the National Natural Science Foundation of China, the Natural Science Foundation of Guangdong Province and the State Key Laboratory of Coordination Chemistry in Nanjing University for their financial support.
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DNA-binding and photocleavage studies of cobalt(III) polypyridyl complexes: [Co(phen) 2 IP] 31 and [Co(phen) 2 PIP] 31 Qian-Ling Zhang, Jin-Gang Liu, Hui Chao, Gen-Qiang Xue, Liang-Nian Ji* Department of Chemistry, Zhongshan University, Guangzhou 510275, PR China Received 29 March 2000; received in revised form 9 June 2000; accepted 22 June 2000
Abstract Two complexes of [Co(phen) 2 IP] 31 (IP5imidazo[4,5-f ][1,10]phenanthroline) and [Co(phen) 2 PIP] 31 (PIP52-phenylimidazo[4,5f ][1,10]phenanthroline) have been synthesized and characterized by UV/ VIS, IR, EA and mass spectra. The binding of the two complexes with calf thymus DNA has been investigated by absorption spectroscopy, cyclic voltammetry, viscosity measurements and DNA cleavage assay. The spectroscopic studies together with cyclic voltammetry and viscosity experiments support that both of the complexes bind to CT DNA by intercalation via IP or PIP into the base pairs of DNA. [Co(phen) 2 PIP] 31 binds more avidly to CT DNA than [Co(phen) 2 IP] 31 , which is consistent with the extended planar and p system of PIP. Noticeably, the two complexes have been found to be efficient photosensitisers for strand scissions in plasmid DNA. 2001 Elsevier Science B.V. All rights reserved. Keywords: Cobalt(III) complexes; DNA-binding; Photocleavage
1. Introduction The interaction of transition metal complexes with DNA has been extensively studied in the past few years. Metal complexes of the type [M(LL) 3 ] n1 where LL is either 1,10-phenanthroline (phen) or a modified phen ligand, are particularly attractive species to recognize and cleave DNA [1–4]. In the early 1980s, Barton et al. demonstrated that tris(phenanthroline) complexes of ruthenium(II) display enantiomeric selectivity in binding to DNA, which can be served as spectroscopic probes in solution to distinguish right- and left-handed DNA helices [5]. Then they found that tris(phenanthroline) complexes of cobalt(III) can cleave DNA when irradiated at 254 nm. Furthermore, they conducted the cleavage reactions by using the high stereospecificity of the tris(diphenylphenanthroline) (DIP) metal isomers. The cleavage reaction is also stereospecific. Incubation of pCo1E1 DNA with L-Co(DIP) 31 yielded no 3 appreciable reaction, whereas incubation with DCo(DIP) 31 showed efficient nicking activity. These find3 ings underscore the importance of an intimate association of the metal with the duplex. The high level of recognition of DNA conformation by these chiral inorganic complexes *Corresponding author. Tel.: 186-20-8411-0115; fax: 186-20-84035497. E-mail address: [email protected] (L.-N. Ji).
suggested the powerful application of stereospecificity in DNA drug design [6]. The features common to these complexes are that the molecule has a high affinity for double-stranded DNA and that the molecule also binds a redox-active metal ion cofactor. The ligands or the metal in these complexes can be varied in an easily controlled manner to facilitate an individual application. All the studies reveal that modification of the metal or ligands would lead to subtle or substantial changes in the binding modes, location and affinity [7,8], giving changes to explore various valuable conformation or site-specific DNA probes and potential chemotherapeutical agents. Currently, much attention has been paid to the complexes of Ru(II) [9–15], but the complexes metal ion other than ruthenium(II) have attracted much less attention. We chose to concentrate our work on complexes of cobalt(III), which have the same interesting characteristics and DNA cleaving properties, but have not received as much attention as the Ru(II) systems [6,16–19]. Clearly further studies using various metals to evaluate the effect of intercalated ligand on the DNA binding and cleavage mechanisms are necessary. In this paper, we report the synthesis, characterization of the two complexes [Co(phen) 2 IP] 31 (IP5imidazo[4,5f ][1,10]phenanthroline) and [Co(phen) 2 PIP] 31 (PIP52phenylimidazo[4,5-f ][1,10]phenanthroline), in which PIP possesses a greater planar area and extended p system than that of IP, and their DNA-binding properties are revealed
0162-0134 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0162-0134( 00 )00132-X
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50
by cyclic voltammetry, electronic absorption, luminescent spectra and viscosity measurements. The photochemical DNA cleavage of the two complexes is also demonstrated. These studies are necessary for the further comprehension of binding of transition metal complexes to DNA, they also can be served as complementary studies for the corresponding complexes of ruthenium.
2. Experimental
2.1. Materials and methods All materials were purchased and used without further purification unless otherwise noted. IP and PIP [20], cis[Co(phen) 2 Cl 2 ]Cl?3H 2 O [21] and [Co(phen) 3 ]Cl 3 [22] were prepared by the methods described previously. The structures of ligands are shown in Fig. 1. All the experiments involving the interaction of the complexes with DNA were carried out in doubly distilled buffer (5 mM Tris–HCl, 50 mM NaCl, pH 7.2). A solution of calf thymus DNA in the buffer gave a ratio of UV absorbance at 260 and 280 nm of about 1.8–1.9:1, indicating that the DNA was sufficiently free of protein [23]. The DNA
concentration per nucleotide was determined by absorption spectroscopy using the molar absorption coefficient (6600 M 21 cm 21 ) at 260 nm [24].
2.1.1. Synthesis of [ Co( phen)2 IP]( ClO4 )3?2 H2 O A mixture of [Co(phen) 2 Cl 2 ]Cl (0.578 g, 1.0 mmol) and IP (0.33 g, 1.5 mmol) in ethanol (50 ml) was refluxed for about 3 h. After filtration, the complex was precipitated upon addition of a saturated ethanolic solution of NaClO 4 . The complex was filtered and further dried under vacuum before recrystallization (acetone–ether). Yield: 0.432 g, 44%. (Found: C, 45.45; H, 3.06; N, 11.83. Calc. for C 37 N 8 H 28 Cl 3 O 14 Co: C, 45.61; H, 2.88; N, 11.51%). lmax / 21 21 nm (´ / M cm ) (water). 272 (44 400), 260(sh), 220 (69 650). nmax / cm 21 , 3446 w(br), 3058 w, 1609 m, 1468 m, 1371 m, 1090 vs, 810 m, 782 s, 706 m and 630 s. FAB-MS: m /z5838 (M-ClO 4 ), 738 (M-2ClO 4 ) and 639 (M-3ClO 4 ). 2.1.2. Synthesis of [ Co( phen)2 PIP]( ClO4 )3?5 H2 O [Co(phen) 2 PIP](ClO 4 ) 3 ?5H 2 O was prepared in a similar way to that of [Co(phen) 2 IP](ClO 4 ) 3 ?2H 2 O. Yield: 0.583 g, 52.8%. (Found: C, 46.49; H, 3.59; N, 10.50. Calc. for C 43 N 8 H 38 Cl 3 O 17 Co: C, 46.76; H, 3.44; N, 10.15%). lmax / 21 21 nm (´ / M cm ) (water). 280 (44 700), 218 (64 850). 21 nmax / cm , 3416 w(br), 3057 w, 1609 m, 1468 m, 1370 m, 1082 vs, 807 m, 779 s, 704 m and 632 s. FAB-MS: m /z5915 (M-ClO 4 ), 814 (M-2ClO 4 ) and 715 (M-3ClO 4 ). Caution: Perchlorate salts of metal complexes with organic ligands are potentially explosive. Only small amounts of the material should be prepared and handled with great care. 2.2. Physical measurements
Fig. 1. Chemical structures of the ligands.
Elemental analyses (C, H and N) were performed on a Perkin-Elmer 240 Q elemental analyser. Infrared spectra were obtained on a Nicolet 170X-FTIR spectrometer as KBr discs. UV-VIS spectra were recorded on a Shimadzu MPS-2000 spectrophotometer. Fast atomic bombardment mass spectra (FAB-MS) were obtained on a VG ZAB-HS spectrometer in a 3-nitrobenzyl alcohol matrix. Cyclic voltammetry was performed on an EGandG PAR 273 polarographic analyser and 270 universal programmer. The supporting electrolyte was 50 mM NaCl, 10 mM Tris, pH 7.2. All samples were purged with nitrogen prior to measurements. A standard three-electrode system was used comprising an Au working electrode, platinum-wire auxiliary electrode and a saturated calomel reference electrode (SCE). For the absorption spectra, equal solution of DNA was added to both complex solution and reference solution to eliminate the absorbance of DNA itself. Viscosity experiments were carried on an Ubbelohde viscometer, immersed in a thermostatted water-bath main-
Q.-L. Zhang et al. / Journal of Inorganic Biochemistry 83 (2001) 49 – 55
tained at 3060.18C. DNA samples approximately 200 base pairs in average length were prepared by sonication in order to mimimize complexities arising from DNA flexibility [25]. Data were presented as (h /h0 )1 / 3 versus the concentration of Co(III) complexes, where h is the viscosity of DNA in the presence of complex, and h0 is the viscosity of DNA alone. Viscosity values were calculated from the observed flow time of DNA-containing solutions (t.100 s) corrected for the flow time of buffer alone (t 0 ), h 5 t 2 t 0 [26]. For the gel electrophoresis experiments, supercoiled pBR 322 DNA (100 mM) was treated with Co(III) complexes in 50 mM Tris–HCl, 18 mM NaCl buffer, pH 7.2, and the solutions were then irradiated at room temperature with a UV lamp (302 nm, 10 W). The samples were analysed by electrophoresis for 2.5 h at 40 V on a 0.8% agarose gel in Tris–acetic acid–EDTA buffer, pH 7.2. The gel was stained with 1 mg / ml ethidium bromide and photographed under UV light.
3. Results and discussion
51
Epa , is 0.095 V, in the absence of DNA. The presence of DNA in the solution at the same concentration of [Co(phen) 2 IP] 31 causes a considerable decrease in the voltammetric current coupled with a slight shift in the E1 / 2 (E1 / 2 50.077 V) to less negative potential (Fig. 2B). For the CV behavior of [Co(phen) 2 PIP] 31 under similar conditions, in the absence of DNA, Epc 5 20.05 V and Epa 50.08 V. The formal potential E1 / 2 is 0.015 V. Upon addition of CT DNA, both the anodic and cathodic peak potential shift to some more negative values (E1 / 2 5 2 0.032 V) coupled with the decrease of its voltammetric current, which is similar to that of [Co(phen) 2 IP] 31 observed, except that the magnitude of the voltammetric current decrease is some more appreciable than that of the [Co(phen) 2 IP] 31 . The drop of the voltammetric currents in the presence of CT DNA can be attributed to diffusion of the metal complex bound to the large, slowly diffusing DNA molecule. The more pronounced decrease of the peak currents upon addition of CT DNA, which are observed for [Co(phen) 2 PIP] 31 over that of [Co(phen) 2 IP] 31 , may indicate the binding affinity of the former to DNA is higher than that of the latter.
3.1. Cyclic voltammetry Typical cyclic voltammetric (CV) behavior of 0.1 mM [Co(phen) 2 IP] 31 in the absence and presence of CT DNA is shown in Fig. 2. The cyclic voltammogram of [Co(phen) 2 IP] 31 in the absence of DNA (Fig. 2A) featured reduction of 31 to the 21 form at a cathodic peak potential, Epc of 0.04 V versus SCE. Reoxidation of 21 occurred, upon scan reversal, at 0.15 V. The separation of the anodic and cathodic peak potentials, DEp 5110 mV, indicated a irreversible redox process. The formal potential E 0 9 (or voltammetric E1 / 2 ), taken as the average of Epc and
Fig. 2. Cyclic voltammograms of [Co(phen) 2 IP] 31 in the absence (A) and presence (B) of DNA in 50 mM NaCl, 10 mM Tris, pH 7.2. [Co]5100 mM, [DNA] / [Co]520. Scan rates, 200 mV s 21 .
3.2. Electronic absorption spectra The application of electronic absorption spectroscopy in DNA-binding studies is one of the most useful techniques [27–29]. Complex binding with DNA through intercalation usually results in hypochromism and bathchromism, due to the intercalative mode involving a strong stacking interaction between an aromatic chromophore and the base pairs of DNA. The extent of the hypochromism commonly parallels the intercalative binding strength. The absorption spectra of the complexes in the absence and presence of calf thymus DNA are illustrated in Fig. 3. The electronic absorption spectra of the two complexes in Tris buffer are similar in shape to that of [Co(phen) 3 ] 31 . In the UV region, the intense absorption bands observed in the Co(III) complexes are attributed to intraligand p2p* transition of the coordinated groups. Addition of increasing amounts of CT DNA results in hypochromism and moderate bathochromic shift in the UV spectra of both [Co(phen) 2 IP] 31 and [Co(phen) 2 PIP] 31 . According to the data presented in Table 1, it seems that the spectral perturbation of the three complexes upon addition of DNA follows: [Co(phen) 2 PIP] 31 . [Co(phen) 2 IP] 31 . [Co(phen) 3 ] 31 . These spectral characteristics may suggest a mode of binding that involves a stacking interaction between the complex and the base pairs of DNA. In order to quantitatively compare the binding strength of the two complexes, the intrinsic binding constants K of the two complexes with CT-DNA were determined according to the following equation [30] through a plot of [DNA] /(´a 2 ´f ) versus [DNA].
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Fig. 3. Absorption spectra of [Co(phen) 2 IP] 31 (A) and [Co(phen) 2 PIP] 31 (B) in the absence (———) and presence (- - -) of DNA with subtraction of the DNA absorbance. [Co]520 mM, [DNA] / [Co]55, 10, 15. Arrow shows the absorbance changes upon increasing DNA concentrations. Insets: plots of [DNA] /(´a 2 ´f ) versus [DNA] for the titration of DNA with complexes; (d) experimental data points; solid line, linear fitting of the data.
[DNA] /(´a 2 ´f ) 5 [DNA] /(´b 2 ´f ) 1 1 /(K(´b 2 ´f )) Where [DNA] is the concentration of DNA in base pairs, the apparent absorption coefficient ´a , ´f and ´b correspond to A obsd / [Co], the extinction coefficient for the free cobalt complex and the extinction coefficient for the cobalt complex in the fully bound form, respectively. In plots [DNA] /(´a 2 ´f ) versus [DNA], K is given by the ratio of slope to intercept. Intrinsic binding constants K of [Co(phen) 2 IP] 31 and [Co(phen) 2 PIP] 31 were obtained about 1.3360.1310 5 and 2.1560.2310 5 M 21 from the decay of the absorbance, respectively, and the binding sites are 4 and 6, respectively. The binding constants indicate that [Co(phen) 2 PIP] 31 binds more strongly than [Co(phen) 2 IP] 31 . This result is expected, since PIP posses-
Table 1 Absorption spectroscopic properties of the Co(III) complexes on binding to DNA Complex
[Co(phen) 2 IP] 31 [Co(phen) 2 PIP] 31 [Co(phen) 3 ] 31 b a b
Absorption lmax (nm) Free
Bound a
220 272 218 280 219 274
223 274 223 282 223 276
[Co]520 mM at [DNA] / [Co]515. From Ref. [19].
Dl
Hypochromicity (%)
3 2 5 2 4 2
19.9 27.9 41.6 28.3 9.6 15.2
ses a greater planar area and extended p system than that of IP, which will lead to PIP penetrating more deeply into, and stacking more strongly with the base pairs of DNA.
3.3. Fluorescence spectroscopic studies The complexes [Co(phen) 2 IP] 31 and [Co(phen) 2 PIP] 31 can emit luminescence in Tris buffer at ambient temperature with maxima at 409 and 410 nm, respectively. Binding of both complexes to DNA was found to increase the fluorescence intensity. The emission spectra of both complexes in the absence and presence of CT DNA are shown in Fig. 4. The plots of the relative intensity versus the ratio of [DNA] / [Co] are also inserted in Fig. 4. Upon addition of CT DNA, the emission intensity increases steadily and reaches 1.4 times larger than that of in the absence of DNA for [Co(phen) 2 IP] 31 , and 2.0 times for [Co(phen) 2 PIP] 31 , respectively, at the ratio of [DNA] / [Co]520. The extent of enhancement increases on going from [Co(phen) 2 IP] 31 to [Co(phen) 2 PIP] 31 , which is consistent with the above absorption spectra results.
3.4. Viscosity studies Further clarification of the interaction between the two complexes and DNA was carried out by viscosity measurements. Optical photophysical probes are necessary, but not sufficient clues to support a binding model. Hydrodynamic measurements that sensitive to length change (i.e., viscosity and sedimentation) are regarded as the least ambiguous
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ligand could bind (or kink) the DNA helix, reduce its effective length and, concomitantly, its viscosity [26,31]. The effects of both complexes and [Co(phen) 3 ] 31 on the viscosity of rod-like DNA are shown in Fig. 5. For [Co(phen) 2 IP] 31 and [Co(phen) 2 PIP] 31 , the viscosity of DNA increases with the increase of the concentration of the complexes, which is similar to that of proven DNA intercalator [Ru(phen) 2 DPPZ] 21 [32]. Both complexes increase the relative viscosity of DNA in a manner consistent with binding by classical intercalation. This result also parallels the pronounced hypochromism and spectral red shift and emission enhancement of both complexes, whereas [Co(phen) 3 ] 31 does not extend DNA helix length. On the basis of the viscosity results, it seems that [Co(phen) 3 ] 31 binds with DNA through groove binding. Although Barton and co-workers have proposed that [Co(phen) 3 ] 31 intercalated into double-stranded DNA [33], this viscosity experiment shows that [Co(phen) 3 ] 31 is not a DNA intercalation agent.
3.5. Photoactivated cleavage of pBR 322 DNA by Co( III) complexes There has been considerable interest in DNA endonucleolytic cleavage reactions that are activated by metal ions [34,35]. The delivery of high concentrations of metal ion to the helix, in locally generating oxygen or hydroxide radicals, yields an efficient DNA cleavage reaction. DNA cleavage was monitored by relation of supercoiled circular pBR 322 (Form I) into nicked circular (Form II) and linear (Form III). When circular plasmid DNA is subjected to electrophoresis, relatively fast migration will be observed
Fig. 4. Emission spectra of [Co(phen) 2 IP] 31 (A) and [Co(phen) 2 PIP] 31 (B) in aqueous buffer (Tris 5 mM, NaCl 50 mM, pH 7.2) at 298 K in the presence of CT DNA. [Co]520 mM, [DNA] / [Co]50, 5, 10, 15, 20. lex 5320 nm. Arrow shows the intensity changes upon increasing concentration. Inset: plots of relative integrated emission intensity versus [DNA] / [Co].
and the most critical tests of binding in solution in the absence of crystallographic structural data [26,31]. A classical intercalation model results in lengthening the DNA helix, as base pairs are separated to accommodate the binding ligand, leading to the increase of DNA viscosity. In contrast, a partial and / or non-classical intercalation of
Fig. 5. Effect of increasing amounts of [Co(phen) 2 IP] 31 (j), [Co(phen) 2 PIP] 31 (d) and [Co(phen) 3 ] 31 (m) on the relative viscosities of CT DNA at 3060.18C.
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for the supercoiled form (Form I). If scission occurs on one strand (nicking), the supercoils will relax to generate a slower-moving open circular form (Form II) [6]. If both strands are cleaved, a linear form (Form III) will be generated that migrates between Forms I and II. Fig. 6 shows the gel electrophoretic separations of plasmid pBR
322 DNA after incubation with Co(III) complexes and irradiation at 302 nm. Fig. 6A,B reveals the conversion of Form I to II after 60 min irradiation in the presence of varying concentrations of [Co(phen) 2 IP] 31 and 31 [Co(phen) 2 PIP] . It can be seen that with increasing the concentration of [Co(phen) 2 IP] 31 and [Co(phen) 2 PIP] 31 , Form II increases gradually, while Form I diminishes gradually. With increasing irradiation time, Form I of pBR 322 DNA diminishes gradually, whereas the amount of Form II increases (Fig. 6C). This is the result of singlestranded cleavage of pBR 322 DNA. It can also be seen in Fig. 6C that neither irradiation of DNA at 302 nm without Co(III) nor incubation with Co(III) without light yields significant strand scission. It is likely that the reduction of Co(III) is the important step leading to DNA cleavage. Further study is necessary to clarify the reaction mechanism.
4. Conclusions Two complexes [Co(phen) 2 IP] 31 and [Co(phen) 2 PIP] 31 are synthesized and characterized. Spectroscopic studies together with cyclic voltammetry and viscosity experiments support that both of the complexes bind to CT DNA by intercalation via IP or PIP into the base pairs of DNA. The intrinsic binding constants indicate that [Co(phen) 2 PIP] 31 binds more avidly to CT DNA than [Co(phen) 2 IP] 31 , which is consistent with the extended planar and p system of PIP. Noticeably, both complexes have been found to promote cleavage of plasmid pBR 322 DNA from the supercoiled form I to the open circular form II upon irradiation, which may be taken as the potential DNA cleavage reagent.
5. Abbreviations CT DNA IL IP phen PIP SCE Fig. 6. (A) Photoactivated cleavage of pBR 322 DNA in the presence of [Co(phen) 2 IP] 31 and light after 60 min irradiation at 302 nm. DNA alone (lane 0), the concentration of [Co(phen) 2 IP] 31 was 10, 20, 30, 40, 50 and 60 mM (lanes 1–6, left to right). (B) Photoactivated cleavage of pBR 322 DNA in the presence of [Co(phen) 2 PIP] 31 and light after 60 min irradiation at 302 nm. The concentration of [Co(phen) 2 PIP] 31 was 10, 20, 40, 50 and 60 mM (lanes 1–5, left to right). (C) Photoactivated cleavage of pBR 322 in the presence of [Co(phen) 2 IP] 31 (20 mM) after irradiation at 302 nm for 0, 20, 40, 60, 80, 100 and 120 min (lanes 1–7, left to right), DNA alone (lane 0).
Calf thymus DNA Intraligand Imidazo[4,5-f ][1,10]phenanthroline 1,10-Phenanthroline 2-Phenylimidazo[4,5-f ][1,10]phenanthroline Saturated calomel electrode
Acknowledgements We are grateful to the National Natural Science Foundation of China, the Natural Science Foundation of Guangdong Province and the State Key Laboratory of Coordination Chemistry in Nanjing University for their financial support.
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