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Effect of intramolecular hydrogen-bond on the DNA-binding and photocleavage properties of polypyridyl cobalt(III) complexes
Inorganica Chimica Acta 339 (2002) 34 /40 www.elsevier.com/locate/ica
Effect of intramolecular hydrogen-bond on the DNA-binding and photocleavage properties of polypyridyl cobalt(III) complexes Qian-Ling Zhang a,b, Jin-Gang Liu c, Jian-Zhong Liu a, Hong Li a, Yi Yang a, Hong Xu a, Hui Chao a, Liang-Nian Ji a,* a
The Key Laboratory of Gene Engineering of Ministry of Education, The State Key Laboratory of Ultrafast Laser Spectroscopy and Department of Chemistry, Zhongshan University, Guangzhou 510275, PR China b Department of Chemistry and Biology, Normal College, Shenzhen University, Shenzhen 518060, PR China c Department of Chemistry, Tongji University, Shanghai 200092, PR China Received 29 August 2001; accepted 25 September 2001 In honor of Professor Helmut Sigel
Abstract Two polypyridyl cobalt(III) complexes containing intramolecular hydrogen-bond [Co(phen)2HPIP]3 (HPIP/2-(2-hydroxyphenyl)imidazo[4,5-f ][1,10]phenanthroline) and [Co(phen)2HNAIP]3 (HNAIP /2-(2-hydroxy-l-naphthyl)imidazo[4,5-f ][1,10]phenanthroline) have been synthesized and characterized by UV /Vis, EA and MS spectra. Binding of both complexes with calf thymus DNA has been investigated by cyclic voltammetry, absorption spectroscopy, luminescence titration, viscosity measurements and DNA cleavage assay. The results suggested that the complex [Co(phen)2HPIP]3 bound to CT DNA by intercalation via HPIP into the base pairs of DNA, while [Co(phen)2HNAIP]3 bound with DNA by partial intercalating the ligand HNAIP. In addition, both complexes have been found to promote the single-stranded cleavage of plasmid pBR 322 DNA upon irradiation, and [Co(phen)2HPIP]3 exhibited the higher cleaving efficiency. # 2002 Published by Elsevier Science B.V. Keywords: DNA-binding; Cobalt complexes; Polypyridyl complexes
1. Introduction There has been substantial interest in understanding the DNA binding properties of transitional metal polypyridyl complexes in the hope of developing novel probes of nucleic acid structures, DNA-cleaving agents, and antitumor drugs [1 /12]. By comparing to the other complexes, the octahedral polypyridyl complexes are particularly suitable for these applications, because they are coordinatively saturated, inert to substitution, rigid and structure well-defined. In addition, these complexes can interact non-covalently with nucleic acids by intercalation, groove binding or electrostatic binding. When the size, shape and chirality of the complexes are fit to the DNA structure, the complexes may approach closely to and intercalate into the base pairs of DNA, and the * Corresponding author. Fax: /86-20-840 35497. E-mail address: [email protected] (L.-N. Ji).
spectroscopic properties such as electronic absorption, steady-state emission, and the thermodynamic properties such as viscosity will experience evident change. On the other hand, upon irradiation, theses complexes can induce the single or double-stranded cleavage of plasmid DNA. Although the interactions of the complexes with DNA have been investigated extensively, the knowledge of the nature of binding of some complexes and their binding geometries has remained relatively modest [8,9,13 /16]. In our group, much effort has been devoted to studying the interactions of novel polypyridyl complexes containing different intercalative ligands with DNA [17 /21]. More recently, we reported the cobalt(III) polypyridyl complex [Co(phen)2PIP]3 (PIP /2phenyl-imidazo[4,5-f ][1,10]phenanthroline) [22], in which the PIP molecule is almost coplanar [23]. Which can interact with DNA by intercalation, and promote the single-stranded cleavage of plasmid DNA upon
0020-1693/02/$ - see front matter # 2002 Published by Elsevier Science B.V. PII: S 0 0 2 0 - 1 6 9 3 ( 0 2 ) 0 0 9 2 3 - 4
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irradiation. Based on PIP, herein we introduce an ortho group to the 2-position of the phenyl group of PIP to form HPIP (HPIP /2-(2-hydroxyphenyl)imidazo[4,5f][1,10]phenanthroline), in which the ortho may form an intramolecular hydrogen-bond with the nitrogen atom of imidazole to extend the planarity. Furthermore, we introduce a phenyl group to HPIP to form HNAIP (HNAIP /2-(2-hydroxy-l-naphthyl)imidazo[4,5-f][1,10] phenanthroline) to further extend the planarity of the ligand. The cobalt(III) complexes of [Co(phen)2HPIP]3 and [Co(phen)2HNAIP]3 (Scheme 1) were also synthesized and their binding properties to calf thymus DNA were studied using absorption spectroscopy, fluorescence spectroscopy, cyclic voltammetry, and viscosity measurements. Their photocleavage behavior toward pBR 322 DNA and the cleaving mechanisms were also investigated.
2. Experimental 2.1. Materials All reagents and solvents were purchased commercially and used without further purification unless otherwise noted. HPIP, HNAIP [24,25] and compound cis -[Co(phen)2Cl2]Cl ×/3H2O [26] were prepared according to the method of our previous study and the literature, respectively. 2.2. Methods and instrumentation All experiments involving the interaction of the complexes with DNA were carried out in twice 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 [27]. The DNA concentration per nucleotide was determined by absorption spectroscopy using the molar absorption coefficient (6600 M1 cm 1) at 260 nm [28]. Elemental analyzes (C, H and N) were carried out on a Perkin /Elmer 240 Q elemental analyzer. Fast atomic bombardment mass spectra (FAB-MS) were obtained on a VG ZAB-HS spectrometer in a 3-nitrobenzyl alcohol matrix. UV /Vis spectra were recorded on a Shimadzu MPS-2000 spectrophotometer and emission
Scheme 1. Chemical structures of the complexes.
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spectra on a Hitachi F-4500 Fluorescence spectrofluorophotometer. For the absorption spectra, equal solution of DNA was added to both complex solution and reference solution to eliminate the absorbance of DNA itself. Cyclic voltammetry was performed on an EG&G PAR 273 polarographic analyzer 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 threeelectrode system was used comprising an Au working electrode, platinum-wire auxiliary electrode and a saturated calomel reference electrode (SCE). Viscosity experiments were carried on an Ubbelodhe viscometer, immersed in a thermostated water-bath maintained at a constant temperature at 30(9/0.1) 8C. DNA samples approximately 200 base pairs in average length were prepared by sonication in order to minimize complexities arising from DNA flexibility [29]. Flow time was measured with a digital stopwatch and each sample was measured three times and an average flow time was calculated. Data were presented as (h /h0)1/3 versus binding ratio [30], 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 (t0), h /t/t0. For the gel electrophorisis experiments, supercoiled pBR 322 DNA (0.1 mg) was treated with Co(III) complexes in 50 mM Tris /HCl, 18 mM NaCl buffer, pH 8.3, and the solution were then irradiated at room temperature (r.t.) with a UV lamp (302 nm, 10 W). The samples were analyzed by electrophorisis for 3 h at 25 V on a 1% agarose gel in tris /acetic acid /EDTA buffer. The gel was stained with 1 mg ml 1 ethidium bromide and photographed under UV light. 2.3. Syntheses 2.3.1. [Co(phen)2(HPIP)](ClO4)3 ×/2H2O A mixture of cis -[Co(phen)2Cl2]Cl ×/3H2O (0.578 g, 1.0 mmol) and HPIP (0.468 g, 1.5 mmol) in EtOH (50 ml) was refluxed for about 3 h. After filtration, the solution was treated with saturated ethanolic solution of NaClO4, and gave a yellow precipitate and further dried under vacuum. The product was further purified by recrystallization from C3H6O /ether. Yield: 78%. Anal. Calc. for [Co(phen)2(HPIP)](ClO4)3 ×/2H2O: C, 48.43; H, 3.00; N, 10.51. Found: C, 48.54; H, 2.94; N, 10.89%. FAB-MS: m /z /930, (M/ClO4), 831, (M/2ClO4) and 731, (M/3ClO4). 2.3.2. [Co(phen)2(HNAIP)](ClO4)3 ×/5H2O This complex was prepared following a method similar to that described for
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[Co(phen)2(HPIP)](ClO4)3 ×/2H2O, with HNAIP in place of HPIP. The purification was also carried out by recrystallization. Yield: 72%. Anal. Calc. for [Co(phen)2(HNAIP)](ClO4)3 ×/5H2O: C, 46.01; H, 3.57; N, 9.99. Found: C, 46.35; H, 3.62; N, 9.87%. FAB-MS: m /z/931, (M/ClO4), 832, (M/2ClO4) and 732, (M/ 3ClO4). 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.
3. Results and discussion
3.1. Electronic absorption spectra Binding of complexes with DNA by intercalation usually results in hypochromism and red shift, due to the intercalative mode involving a strong stacking interaction between the aromatic chromophore and the base pairs of DNA. The magnitude of the hypochromism and red shift are commonly found to depend on the strength of the intercalative interaction [31]. The absorption spectra of [Co(phen)2(HPIP)]3 and [Co(phen)2(HNAIP)]3 (at constant concentration of complex) in the presence of increasing amounts of calf thymus DNA was shown in Fig. 1. From Fig. 1, it can be seen that both complexes exhibited intense absorption bands in the UV region, which are attributed to intraligand (IL) p/p* transition of the coordinated groups, and addition of increasing amounts of CT DNA resulted in hypochromism and bathocromic shift in the UV spectra of both complexes. The hypochromism in the IL band reached as high as 47.1% at 274 nm with 7 nm red shift for [Co(phen)2(HPIP)]3. The corresponding band in [Co(phen)2(HNAIP)]3 at 273 nm exhibited hypochromism about 25% and a bathochromic shift of 3 nm under similar experimental conditions. These spectral characteristics suggest that both the complexes bind to DNA by intercalation, and
Fig. 1. Absorption spectra of [Co(phen)2(HPIP)]3 (A) and [Co(phen)2(HNAIP)]3 (B) in the absence ( */) and presence (---) of DNA with subtraction of the DNA absorbance. [Co]/20 mM.
the complex [Co(phen)2(HPIP)]3 binds to DNA more strongly than [Co(phen)2(HNAIP)]3. In order to quantitatively compare the binding strength of the two complexes, the intrinsic binding constants K of the complexes with CT-DNA were calculated using the following function equation [32]: [DNA]=(o a o f )[DNA]=(o b o f )1=(K(o b o f )) where [DNA] is the concentration of DNA in base pairs, the apparent absorption coefficient o a, o f and o b correspond to Aobsd/[Co], the extinction coefficient for the free cobalt complex and the extinction coefficient for the free cobalt complex in the fully bound form, respectively. In plots [DNA]/(o a/o f) vs [DNA], K is given by the ratio of slope to the y intercept. The intrinsic binding constants K of [Co(phen)2(HPIP)]3 and [Co(phen)2(HNAIP)]3 were determined to be 4.1 / 105 and 1.8 /105 M 1, respectively. Comparing to their parent complex [Co(phen)2(PIP)]3 (PIP /2-phenylimidazo[4,5-f][1,10]phenanthroline), whose K is 2.2 /105 M 1 [22], the binding constants of the complexes were in the series of [Co(phen)2(HNAIP)]3 B/ 3 [Co(phen)2(PIP)] B/[Co(phen)2(HPIP)]3. The stronger binding affinity of [Co(phen)2(HPIP)]3 may be explained by the fact that in this complex, the ortho group of HPIP would be considered to be almost coplanar with the imidazole ring owing to forming an intramolecular hydrogen bond with the nitrogen atom of the imidazole ring [33], which have also been seen with analogous compounds in their crystal structures [34 /38]. While for complex [Co(phen)2(HNAIP)]3, which exhibited the lowest binding affinity to DNA, it is likely that the large naphthyl ring of HNAIP may lie out of the plane of the imidazole ring, thus the complex cannot intercalate completely into the DNA base pairs, and concomitantly, decreasing the DNA-binding affinity of complex. 3.2. Emission spectra The complexes [Co(phen)2(HPIP)]3 and 3 [Co(phen)2(HNAIP)] can emit luminescence in Tris buffer at ambient temperature. Despite the structural similarity of the two complexes, their interactions with double-strand calf thymus DNA are somewhat different, as revealed by the luminescence behavior in the presence of DNA. The emission spectra of [Co(phen)2(HPIP)]3 in the presence of increasing amounts of DNA as given in Fig. 2. After binding to CT DNA, the emission intensity of [Co(phen)2(HPIP)]3 increased sharply and reaches as high as 8.2 times that of in the absence of DNA. This indicates that [Co(phen)2(HPIP)]3 binds with DNA by intercalation, and such enhancement could be ascribed to the fact that the intercalation of the complex into DNA, which is hydrophobic inside the helix, limits the
Q.-L. Zhang et al. / Inorganica Chimica Acta 339 (2002) 34 /40
Fig. 2. Emission spectra of [Co(phen)2(HPIP)]3 in aqueous buffer (5 mM Tris, 50 mM NaCl, pH 7.2) at 298 K in the presence of CT DNA. [Co]/20 mM, lex /300 nm.
collision and energy dissipation with the environmental water and oxygen. While for the complex [Co(phen)2(HNAIP)]3, the emission enhancement extent is less pronounced than that of [Co(phen)2(HPIP)]3, implying that [Co(phen)2(HNAIP)]3 has a much lower binding affinity to DNA. These results also indicate that [Co(phen)2(HPIP)]3 binds to DNA by an intercalative mode and exhibits the stronger binding affinity, while [Co(phen)2(HNAIP)]3 binds to DNA by a partial intercalating mode and has the lower binding affinity. 3.3. Cyclic voltammetry The application of electrochemical methods to the study of metallointercalation and coordination of transitional metal complexes to DNA provides a useful complement to the previously used methods of investigation, such as UV /Vis spectroscopy. The cyclic voltammetric behavior of the two complexes in the presence of CT DNA is shown in Table 1. As seen in Table 1, the cyclic voltammetric data of the two complexes in the absence of DNA featured the reduction of 3/ to the 2/ form at a cathodic peak potential, Epc of 0.180 V for [Co(phen)2(HPIP)]3 and 0.188 V for [Co(phen)2(HNAIP)]3 vs SCE. And the reoxidation of the 2/ form occurred at 0.088 and 0.098 V, respectively, upon scan reversal. In the absence of DNA, the separation of the anodic and cathodic peak potentials, DEp /92 mV for [Co(phen)2(HPIP)]3 and 90 mV for
37
[Co(phen)2(HNAIP)]3, indicating a reversible redox process. The formal potential, E 8? (or voltammetric E1/2), taken as the average of Epc and Epa are 0.134 and 0.143 V, respectively. The presence of DNA in the solution at the same concentration of both complexes caused a considerable decrease in the voltammetric current. In addition, the peak potentials, both Epc and Epa, as well as E1/2 had a shift to less negative potential. These results further indicate that [Co(phen)2(HPIP)]3 and [Co(phen)2(HNAIP)]3 can intercalate into DNA by the HPIP or HNAIP ligands. 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, and the complex [Co(phen)2(HPIP)]3 binds to DNA more strongly than [Co(phen)2(HNAIP)]3. 3.4. Viscosity studies The binding modes of the two complexes were further investigated by viscosity measurements. Photophysical probes generally provide 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 and the most critical tests of binding mode in solution in the absence of crystallographic structural data [39,40]. 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. However, a partial and/or non-classical intercalation of ligand may bend (or kink) DNA helix, resulting in the decrease of its effective length and, concomitantly, its viscosity. The effects of the complexes [Co(phen)2(HPIP)]3 and 3 [Co(phen)2(HNAIP)] , together with [Co(phen)2(PIP)]3 on the viscosity of rod-like DNA are shown in Fig. 3. For the complexes [Co(phen)2(PIP)]3 and [Co(phen)2(HPIP)]3, as increasing the amounts of complexes, the viscosity of DNA increases steadily, which is similar to that of the classical intercalative complex [Ru(phen)2(DPPZ)]2. While for the complex [Co(phen)2(HNAIP)]3, the increasing extent of the viscosity of DNA is less
Table 1 Cyclic voltammetric behavior of complexes in the presence of CT DNA R
Epa (V)
Epc (V)
DEp (mV)
E1/2 (V)
ipa/ipa
[Co(phen)2HPIP]
0 20 40
0.088 0.074 0.064
0.180 0.180 0.178
92 106 114
0.134 0.127 0.121
1.00 1.32 1.48
[Co(phen)2HNAIP]3
0 20 40
0.098 0.080 0.064
0.188 0.180 0.176
90 100 112
0.143 0.130 0.120
1.00 1.30 1.46
Complex 3
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Fig. 4. Photoactivated cleavage of pBR 322 DNA in the presence of complex and light after 60 min irradiation at 302 nm. DNA alone (lane 0), (1 /4) in different concentration of [Co(phen)2(HPIP)]3 . (1) 80; (2) 60; (3) 40; (4) 20 mM. (5 /8) in different concentration of [Co(phen)2(HNAIP)]3 . (5) 80; (6) 60; (7) 40; (8) 20 mM. Fig. 3. Effect of increasing amounts of [Co(phen)2(PIP)]3 ('), [Co(phen)2(HPIP)]3 (j), and [Co(phen)2(HNAIP)]3 (m) on the relative viscosities of CT DNA at 30(9/0.1) 8C, [DNA]/0.5 mM.
pronounced than that of [Co(phen)2(PIP)]3 and [Co(phen)2(HPIP)]3. This indicates that [Co(phen)2(HNAIP)]3 binds to DNA less deeply and strongly than the above two complexes, and it may bind to DNA by a partial intercalation. Based on the above experiments, the results have clearly indicated the different ability of the ligands in the three complexes [Co(phen)2(PIP)]3, 3 [Co(phen)2(HPIP)] and [Co(phen)2(HNAIP)]3, to stack and overlap with the base pairs. Although the crystal structure of these complexes are unavailable at present, in our previous studies, we have got the crystal structure of the ligand PIP, in which the phenyl ring is almost coplanar with the imidazole ring. Herein, we think that the ortho phenolic group of HPIP can also be well coplanar with the imidazole ring due to forming an intramolecular hydrogen-bond with the nitrogen atom of imidazole ring, which have also been seen with analogous compounds in their crystal structures [34 / 38]. The introduction of ortho phenolic group to the HPIP molecule may have some additional affinity to stabilize the complexes binding to DNA. While in the ligand HNAIP, the large naphthyl ring may lie out of the plane of the imidazole ring, decreasing the planarity of ligand, thus resulting in the partial intercalation of the HNAIP ligand. These results parallel to the above absorption spectra and emission spectra of both complexes in the presence of DNA. 3.5. Photocleavage Besides the above methods, the binding of the two complexes to DNA was also investigated by DNA photocleavage assay. Irradiation of complexes [Co(phen)2(HPIP)]3 or [Co(phen)2(HNAIP)]3 and plasmid in tris /acetate buffer (pH 7.2) at 302 nm under aerobic conditions results in the cleavage of supercoiled form I of the plasmid pBR 322 DNA to the nicked form II (Fig. 4). No DNA cleavage was observed for controls in which the complex was absent (Fig. 4, lane 0), or incubation of the plasmid with either complex in dark
(data not presented). As increasing the concentration of [Co(phen)2(HPIP)]3 and [Co(phen)2(HNAIP)]3, the amount of form II increases gradually, while form I diminishes gradually. On the other hand, the complexes exhibited different cleaving efficiency for the plasmid DNA. [Co(phen)2(HPIP)]3 can induce the obvious cleavage of the plasmid DNA at the concentration of 20 mM. However, [Co(phen)2(HNAIP)]3 exhibited much lower cleaving efficiency for DNA. Even at the concentration of 80 mM, [Co(phen)2(HNAIP)]3 cannot promote the complete conversion of DNA from form I to II. The different DNA-cleavage efficiency of the two complexes was due to the different binding affinity of the complexes to DNA. In order to establish the reactive species such as singlet oxygen, superoxide anion radical and hydroxyl radicals that are responsible for the photoactivated cleavage of the plasmid DNA, the following experiments are carried out. For [Co(phen)2(HPIP)]3, firstly, the cleavage was performed in the presence of D2O to test the possibility that the photoactivated cleavage involves the formation of singlet oxygen, which is known to react with guanine residues at neutral pH. Singlet oxygen would be expected to induce more strand scissions in D2O than in H2O, owing to its longer lifetime in the former solvent [41]. As seen in Fig. 5(A), lane 3, no enhancement but inhibition was observed. This indicated that the singlet oxygen was not the active species in the cleavage reaction. Furthermore, studies with the singlet oxygen quencher histidine were carried out [42], and no obvious inhibition was observed (Fig. 5(A), lane 2), which further confirmed that singlet oxygen was not involved in the cleavage. On the other hand, in the + presence of a superoxide anion radical (O2 ) scavenger such as superoxide dismutase (SOD) (1000 u ml1) (Fig. 5(A), lane 4), the cleavage was obviously inhibited, indicating that superoxide anion radical is likely the reactive species. At the same time, in the presence of different hydroxyl radical scavengers such as mannitol [43] (50.0 mM) (Fig. 5(A), lane 5), ethanol (1.7 mM) (Fig. 5(A), lane 6), sodium formate (100.0 mM) (Fig. 5(A), lane 7), and DMSO (10.0 mM) (Fig. 5(A), lane 8), the different degrees of inhibition in the photoinduced
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properties were also studied. Spectroscopic studies together with cyclic voltammetry and viscosity experiments supported that [Co(phen)2(HPIP)]3 bound to DNA by intercalation, and exhibited stronger binding affinity to DNA. While [Co(phen)2(HNAIP)]3 bound to DNA by a partial intercalative mode, and exhibited relatively lower binding affinity. In addition, both complexes have been found to promote the singlestranded cleavage of plasmid pBR 322 DNA upon irradiation. The cleaving mechanisms between complexes and DNA were also proposed.
Acknowledgements
Fig. 5. Photoactivated cleavage of pBR 322 (0.1 mg) in the presence of complex and different inhibitors after irradiation at 302 nm for 60 min. (A) In the presence of 40 mM [Co(phen)2(HPIP)]3 : (0) no complex; (1) no inhibitor; (2) in histidine (1.2 mM); (3) D2O used instead of buffer; (4) in SOD (1000 u ml 1); (5) in mannitol (50.0 mM); (6) in ethanol (1.7 mM); (7) in sodium formate (100.0 mM); (8) in DMSO (10.0 mM). (B) In the presence of 40 mM [Co(phen)2(HNAIP)]3 : (0) no complex; (1) no inhibitor; (2) in histidine (1.2 mM); (3) D2O used instead of buffer; (4) in SOD (1000 u ml 1); (5) in mannitol (50.0 mM); (6) in ethanol (1.7 mM); (7) in sodium formate (100.0 mM); (8) in DMSO (10.0 mM).
cleavage of the plasmid by [Co(phen)2(HPIP)]3 were also observed, suggesting that in addition to the superoxide anion radical, hydroxyl radical is also likely to be one of the reactive species for the cleavage. For the complex [Co(phen)2(HNAIP)]3, the same experiments were also conducted to investigate the reactive species responsible for the cleavage reaction. From Fig. 5(B), it can be seen that in the presence of histidine, no obvious inhibition was observed. Furthermore, no obvious enhancement was observed in D2O (lane 3), this indicated that the singlet oxygen was not the reactive specie in the cleavage. In addition, in the presence of the SOD, the cleavage was also not inhibited, indicating that superoxide anion radical + (O2 ) was not involved in the cleavage. However, in the presence of different hydroxyl radical scavengers such as mannitol (lane 5), ethanol (lane 6), sodium formate (lane 7), and DMSO (lane 8), the different degrees of inhibition in the photoinduced cleavage of the plasmid were observed, this suggested that the hydroxyl radical is likely to be the reactive species for the cleavage reaction.
4. Conclusions Two complexes [Co(phen)2(HPIP)]3 and 3 [Co(phen)2(HNAIP)] have been synthesized and characterized. Their DNA-binding and photocleavage
We are grateful to the National Natural Science Foundation of China, the Natural Science Foundation of Guangdong Province, the State Key Laboratory of Bio-organic and Natural Products Chemistry in Shanghai Institute of Organic Chemistry, the State Key Laboratory of Coordination Chemistry in Nanjing University, and the Visiting Scholar Foundation of Key Laboratory in University for their financial support.
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[32] R.P. Hertzberg, P.B. Dervan, J. Am. Chem. Soc. 104 (1982) 313. [33] J.-Z. Wu, L. Wang, G. Yang, L.-N. Ji, N. Katsaros, A. Koutsodiumou, Cryst. Res. Technol. 31 (1996) 857. [34] P. Ste´nson, Acta Chem. Scand. 24 (1970) 3729. [35] R. Hage, J.G. Haasnoot, J. Reedijk, R. Wang, E.M. Ryan, J.G. Vos, A.L. Spek, A.J.M. Duisenberg, Inorg. Chim. Acta 174 (1990) 77. [36] B.M. Holligan, J.C. Jeffery, M.K. Norgett, E. Schatz, M.D. Ward, J. Chem. Soc., Dalton Trans. (1992) 3345. [37] J.C. Jeffery, C.S.G. Moore, F. Psillakis, M.D. Ward, Polyhedron 14 (1995) 599. [38] E. Shuter, H.R. Hoveyda, V. Karunaratne, S.L. Rettig, C. Orvig, Inorg. Chem. 35 (1996) 368. [39] S. Satyanaryana, J.C. Daborusak, J.B. Chaires, Biochem. 32 (1993) 2573. [40] S. Satyanarayana, J.C. Dabroniak, J.B. Chaires, Biochem. 31 (1992) 9319. [41] A.U. Khan, J. Phys. Chem. 80 (1976) 2219. [42] C.C. Cheng, S.E. Rokita, C.J. Burrows, Angew. Chem., Int. Ed. Engl. 32 (1993) 277. [43] J.M. Kelly, D.J. McConnell, C. OhUigin, A.B. Tossi, A. KirschDe Mesmaeker, A. Masschelein, J. Nasielski, J. Chem. Soc., Chem. Commun. (1987) 1821.
Effect of intramolecular hydrogen-bond on the DNA-binding and photocleavage properties of polypyridyl cobalt(III) complexes Qian-Ling Zhang a,b, Jin-Gang Liu c, Jian-Zhong Liu a, Hong Li a, Yi Yang a, Hong Xu a, Hui Chao a, Liang-Nian Ji a,* a
The Key Laboratory of Gene Engineering of Ministry of Education, The State Key Laboratory of Ultrafast Laser Spectroscopy and Department of Chemistry, Zhongshan University, Guangzhou 510275, PR China b Department of Chemistry and Biology, Normal College, Shenzhen University, Shenzhen 518060, PR China c Department of Chemistry, Tongji University, Shanghai 200092, PR China Received 29 August 2001; accepted 25 September 2001 In honor of Professor Helmut Sigel
Abstract Two polypyridyl cobalt(III) complexes containing intramolecular hydrogen-bond [Co(phen)2HPIP]3 (HPIP/2-(2-hydroxyphenyl)imidazo[4,5-f ][1,10]phenanthroline) and [Co(phen)2HNAIP]3 (HNAIP /2-(2-hydroxy-l-naphthyl)imidazo[4,5-f ][1,10]phenanthroline) have been synthesized and characterized by UV /Vis, EA and MS spectra. Binding of both complexes with calf thymus DNA has been investigated by cyclic voltammetry, absorption spectroscopy, luminescence titration, viscosity measurements and DNA cleavage assay. The results suggested that the complex [Co(phen)2HPIP]3 bound to CT DNA by intercalation via HPIP into the base pairs of DNA, while [Co(phen)2HNAIP]3 bound with DNA by partial intercalating the ligand HNAIP. In addition, both complexes have been found to promote the single-stranded cleavage of plasmid pBR 322 DNA upon irradiation, and [Co(phen)2HPIP]3 exhibited the higher cleaving efficiency. # 2002 Published by Elsevier Science B.V. Keywords: DNA-binding; Cobalt complexes; Polypyridyl complexes
1. Introduction There has been substantial interest in understanding the DNA binding properties of transitional metal polypyridyl complexes in the hope of developing novel probes of nucleic acid structures, DNA-cleaving agents, and antitumor drugs [1 /12]. By comparing to the other complexes, the octahedral polypyridyl complexes are particularly suitable for these applications, because they are coordinatively saturated, inert to substitution, rigid and structure well-defined. In addition, these complexes can interact non-covalently with nucleic acids by intercalation, groove binding or electrostatic binding. When the size, shape and chirality of the complexes are fit to the DNA structure, the complexes may approach closely to and intercalate into the base pairs of DNA, and the * Corresponding author. Fax: /86-20-840 35497. E-mail address: [email protected] (L.-N. Ji).
spectroscopic properties such as electronic absorption, steady-state emission, and the thermodynamic properties such as viscosity will experience evident change. On the other hand, upon irradiation, theses complexes can induce the single or double-stranded cleavage of plasmid DNA. Although the interactions of the complexes with DNA have been investigated extensively, the knowledge of the nature of binding of some complexes and their binding geometries has remained relatively modest [8,9,13 /16]. In our group, much effort has been devoted to studying the interactions of novel polypyridyl complexes containing different intercalative ligands with DNA [17 /21]. More recently, we reported the cobalt(III) polypyridyl complex [Co(phen)2PIP]3 (PIP /2phenyl-imidazo[4,5-f ][1,10]phenanthroline) [22], in which the PIP molecule is almost coplanar [23]. Which can interact with DNA by intercalation, and promote the single-stranded cleavage of plasmid DNA upon
0020-1693/02/$ - see front matter # 2002 Published by Elsevier Science B.V. PII: S 0 0 2 0 - 1 6 9 3 ( 0 2 ) 0 0 9 2 3 - 4
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irradiation. Based on PIP, herein we introduce an ortho group to the 2-position of the phenyl group of PIP to form HPIP (HPIP /2-(2-hydroxyphenyl)imidazo[4,5f][1,10]phenanthroline), in which the ortho may form an intramolecular hydrogen-bond with the nitrogen atom of imidazole to extend the planarity. Furthermore, we introduce a phenyl group to HPIP to form HNAIP (HNAIP /2-(2-hydroxy-l-naphthyl)imidazo[4,5-f][1,10] phenanthroline) to further extend the planarity of the ligand. The cobalt(III) complexes of [Co(phen)2HPIP]3 and [Co(phen)2HNAIP]3 (Scheme 1) were also synthesized and their binding properties to calf thymus DNA were studied using absorption spectroscopy, fluorescence spectroscopy, cyclic voltammetry, and viscosity measurements. Their photocleavage behavior toward pBR 322 DNA and the cleaving mechanisms were also investigated.
2. Experimental 2.1. Materials All reagents and solvents were purchased commercially and used without further purification unless otherwise noted. HPIP, HNAIP [24,25] and compound cis -[Co(phen)2Cl2]Cl ×/3H2O [26] were prepared according to the method of our previous study and the literature, respectively. 2.2. Methods and instrumentation All experiments involving the interaction of the complexes with DNA were carried out in twice 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 [27]. The DNA concentration per nucleotide was determined by absorption spectroscopy using the molar absorption coefficient (6600 M1 cm 1) at 260 nm [28]. Elemental analyzes (C, H and N) were carried out on a Perkin /Elmer 240 Q elemental analyzer. Fast atomic bombardment mass spectra (FAB-MS) were obtained on a VG ZAB-HS spectrometer in a 3-nitrobenzyl alcohol matrix. UV /Vis spectra were recorded on a Shimadzu MPS-2000 spectrophotometer and emission
Scheme 1. Chemical structures of the complexes.
35
spectra on a Hitachi F-4500 Fluorescence spectrofluorophotometer. For the absorption spectra, equal solution of DNA was added to both complex solution and reference solution to eliminate the absorbance of DNA itself. Cyclic voltammetry was performed on an EG&G PAR 273 polarographic analyzer 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 threeelectrode system was used comprising an Au working electrode, platinum-wire auxiliary electrode and a saturated calomel reference electrode (SCE). Viscosity experiments were carried on an Ubbelodhe viscometer, immersed in a thermostated water-bath maintained at a constant temperature at 30(9/0.1) 8C. DNA samples approximately 200 base pairs in average length were prepared by sonication in order to minimize complexities arising from DNA flexibility [29]. Flow time was measured with a digital stopwatch and each sample was measured three times and an average flow time was calculated. Data were presented as (h /h0)1/3 versus binding ratio [30], 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 (t0), h /t/t0. For the gel electrophorisis experiments, supercoiled pBR 322 DNA (0.1 mg) was treated with Co(III) complexes in 50 mM Tris /HCl, 18 mM NaCl buffer, pH 8.3, and the solution were then irradiated at room temperature (r.t.) with a UV lamp (302 nm, 10 W). The samples were analyzed by electrophorisis for 3 h at 25 V on a 1% agarose gel in tris /acetic acid /EDTA buffer. The gel was stained with 1 mg ml 1 ethidium bromide and photographed under UV light. 2.3. Syntheses 2.3.1. [Co(phen)2(HPIP)](ClO4)3 ×/2H2O A mixture of cis -[Co(phen)2Cl2]Cl ×/3H2O (0.578 g, 1.0 mmol) and HPIP (0.468 g, 1.5 mmol) in EtOH (50 ml) was refluxed for about 3 h. After filtration, the solution was treated with saturated ethanolic solution of NaClO4, and gave a yellow precipitate and further dried under vacuum. The product was further purified by recrystallization from C3H6O /ether. Yield: 78%. Anal. Calc. for [Co(phen)2(HPIP)](ClO4)3 ×/2H2O: C, 48.43; H, 3.00; N, 10.51. Found: C, 48.54; H, 2.94; N, 10.89%. FAB-MS: m /z /930, (M/ClO4), 831, (M/2ClO4) and 731, (M/3ClO4). 2.3.2. [Co(phen)2(HNAIP)](ClO4)3 ×/5H2O This complex was prepared following a method similar to that described for
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[Co(phen)2(HPIP)](ClO4)3 ×/2H2O, with HNAIP in place of HPIP. The purification was also carried out by recrystallization. Yield: 72%. Anal. Calc. for [Co(phen)2(HNAIP)](ClO4)3 ×/5H2O: C, 46.01; H, 3.57; N, 9.99. Found: C, 46.35; H, 3.62; N, 9.87%. FAB-MS: m /z/931, (M/ClO4), 832, (M/2ClO4) and 732, (M/ 3ClO4). 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.
3. Results and discussion
3.1. Electronic absorption spectra Binding of complexes with DNA by intercalation usually results in hypochromism and red shift, due to the intercalative mode involving a strong stacking interaction between the aromatic chromophore and the base pairs of DNA. The magnitude of the hypochromism and red shift are commonly found to depend on the strength of the intercalative interaction [31]. The absorption spectra of [Co(phen)2(HPIP)]3 and [Co(phen)2(HNAIP)]3 (at constant concentration of complex) in the presence of increasing amounts of calf thymus DNA was shown in Fig. 1. From Fig. 1, it can be seen that both complexes exhibited intense absorption bands in the UV region, which are attributed to intraligand (IL) p/p* transition of the coordinated groups, and addition of increasing amounts of CT DNA resulted in hypochromism and bathocromic shift in the UV spectra of both complexes. The hypochromism in the IL band reached as high as 47.1% at 274 nm with 7 nm red shift for [Co(phen)2(HPIP)]3. The corresponding band in [Co(phen)2(HNAIP)]3 at 273 nm exhibited hypochromism about 25% and a bathochromic shift of 3 nm under similar experimental conditions. These spectral characteristics suggest that both the complexes bind to DNA by intercalation, and
Fig. 1. Absorption spectra of [Co(phen)2(HPIP)]3 (A) and [Co(phen)2(HNAIP)]3 (B) in the absence ( */) and presence (---) of DNA with subtraction of the DNA absorbance. [Co]/20 mM.
the complex [Co(phen)2(HPIP)]3 binds to DNA more strongly than [Co(phen)2(HNAIP)]3. In order to quantitatively compare the binding strength of the two complexes, the intrinsic binding constants K of the complexes with CT-DNA were calculated using the following function equation [32]: [DNA]=(o a o f )[DNA]=(o b o f )1=(K(o b o f )) where [DNA] is the concentration of DNA in base pairs, the apparent absorption coefficient o a, o f and o b correspond to Aobsd/[Co], the extinction coefficient for the free cobalt complex and the extinction coefficient for the free cobalt complex in the fully bound form, respectively. In plots [DNA]/(o a/o f) vs [DNA], K is given by the ratio of slope to the y intercept. The intrinsic binding constants K of [Co(phen)2(HPIP)]3 and [Co(phen)2(HNAIP)]3 were determined to be 4.1 / 105 and 1.8 /105 M 1, respectively. Comparing to their parent complex [Co(phen)2(PIP)]3 (PIP /2-phenylimidazo[4,5-f][1,10]phenanthroline), whose K is 2.2 /105 M 1 [22], the binding constants of the complexes were in the series of [Co(phen)2(HNAIP)]3 B/ 3 [Co(phen)2(PIP)] B/[Co(phen)2(HPIP)]3. The stronger binding affinity of [Co(phen)2(HPIP)]3 may be explained by the fact that in this complex, the ortho group of HPIP would be considered to be almost coplanar with the imidazole ring owing to forming an intramolecular hydrogen bond with the nitrogen atom of the imidazole ring [33], which have also been seen with analogous compounds in their crystal structures [34 /38]. While for complex [Co(phen)2(HNAIP)]3, which exhibited the lowest binding affinity to DNA, it is likely that the large naphthyl ring of HNAIP may lie out of the plane of the imidazole ring, thus the complex cannot intercalate completely into the DNA base pairs, and concomitantly, decreasing the DNA-binding affinity of complex. 3.2. Emission spectra The complexes [Co(phen)2(HPIP)]3 and 3 [Co(phen)2(HNAIP)] can emit luminescence in Tris buffer at ambient temperature. Despite the structural similarity of the two complexes, their interactions with double-strand calf thymus DNA are somewhat different, as revealed by the luminescence behavior in the presence of DNA. The emission spectra of [Co(phen)2(HPIP)]3 in the presence of increasing amounts of DNA as given in Fig. 2. After binding to CT DNA, the emission intensity of [Co(phen)2(HPIP)]3 increased sharply and reaches as high as 8.2 times that of in the absence of DNA. This indicates that [Co(phen)2(HPIP)]3 binds with DNA by intercalation, and such enhancement could be ascribed to the fact that the intercalation of the complex into DNA, which is hydrophobic inside the helix, limits the
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Fig. 2. Emission spectra of [Co(phen)2(HPIP)]3 in aqueous buffer (5 mM Tris, 50 mM NaCl, pH 7.2) at 298 K in the presence of CT DNA. [Co]/20 mM, lex /300 nm.
collision and energy dissipation with the environmental water and oxygen. While for the complex [Co(phen)2(HNAIP)]3, the emission enhancement extent is less pronounced than that of [Co(phen)2(HPIP)]3, implying that [Co(phen)2(HNAIP)]3 has a much lower binding affinity to DNA. These results also indicate that [Co(phen)2(HPIP)]3 binds to DNA by an intercalative mode and exhibits the stronger binding affinity, while [Co(phen)2(HNAIP)]3 binds to DNA by a partial intercalating mode and has the lower binding affinity. 3.3. Cyclic voltammetry The application of electrochemical methods to the study of metallointercalation and coordination of transitional metal complexes to DNA provides a useful complement to the previously used methods of investigation, such as UV /Vis spectroscopy. The cyclic voltammetric behavior of the two complexes in the presence of CT DNA is shown in Table 1. As seen in Table 1, the cyclic voltammetric data of the two complexes in the absence of DNA featured the reduction of 3/ to the 2/ form at a cathodic peak potential, Epc of 0.180 V for [Co(phen)2(HPIP)]3 and 0.188 V for [Co(phen)2(HNAIP)]3 vs SCE. And the reoxidation of the 2/ form occurred at 0.088 and 0.098 V, respectively, upon scan reversal. In the absence of DNA, the separation of the anodic and cathodic peak potentials, DEp /92 mV for [Co(phen)2(HPIP)]3 and 90 mV for
37
[Co(phen)2(HNAIP)]3, indicating a reversible redox process. The formal potential, E 8? (or voltammetric E1/2), taken as the average of Epc and Epa are 0.134 and 0.143 V, respectively. The presence of DNA in the solution at the same concentration of both complexes caused a considerable decrease in the voltammetric current. In addition, the peak potentials, both Epc and Epa, as well as E1/2 had a shift to less negative potential. These results further indicate that [Co(phen)2(HPIP)]3 and [Co(phen)2(HNAIP)]3 can intercalate into DNA by the HPIP or HNAIP ligands. 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, and the complex [Co(phen)2(HPIP)]3 binds to DNA more strongly than [Co(phen)2(HNAIP)]3. 3.4. Viscosity studies The binding modes of the two complexes were further investigated by viscosity measurements. Photophysical probes generally provide 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 and the most critical tests of binding mode in solution in the absence of crystallographic structural data [39,40]. 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. However, a partial and/or non-classical intercalation of ligand may bend (or kink) DNA helix, resulting in the decrease of its effective length and, concomitantly, its viscosity. The effects of the complexes [Co(phen)2(HPIP)]3 and 3 [Co(phen)2(HNAIP)] , together with [Co(phen)2(PIP)]3 on the viscosity of rod-like DNA are shown in Fig. 3. For the complexes [Co(phen)2(PIP)]3 and [Co(phen)2(HPIP)]3, as increasing the amounts of complexes, the viscosity of DNA increases steadily, which is similar to that of the classical intercalative complex [Ru(phen)2(DPPZ)]2. While for the complex [Co(phen)2(HNAIP)]3, the increasing extent of the viscosity of DNA is less
Table 1 Cyclic voltammetric behavior of complexes in the presence of CT DNA R
Epa (V)
Epc (V)
DEp (mV)
E1/2 (V)
ipa/ipa
[Co(phen)2HPIP]
0 20 40
0.088 0.074 0.064
0.180 0.180 0.178
92 106 114
0.134 0.127 0.121
1.00 1.32 1.48
[Co(phen)2HNAIP]3
0 20 40
0.098 0.080 0.064
0.188 0.180 0.176
90 100 112
0.143 0.130 0.120
1.00 1.30 1.46
Complex 3
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Fig. 4. Photoactivated cleavage of pBR 322 DNA in the presence of complex and light after 60 min irradiation at 302 nm. DNA alone (lane 0), (1 /4) in different concentration of [Co(phen)2(HPIP)]3 . (1) 80; (2) 60; (3) 40; (4) 20 mM. (5 /8) in different concentration of [Co(phen)2(HNAIP)]3 . (5) 80; (6) 60; (7) 40; (8) 20 mM. Fig. 3. Effect of increasing amounts of [Co(phen)2(PIP)]3 ('), [Co(phen)2(HPIP)]3 (j), and [Co(phen)2(HNAIP)]3 (m) on the relative viscosities of CT DNA at 30(9/0.1) 8C, [DNA]/0.5 mM.
pronounced than that of [Co(phen)2(PIP)]3 and [Co(phen)2(HPIP)]3. This indicates that [Co(phen)2(HNAIP)]3 binds to DNA less deeply and strongly than the above two complexes, and it may bind to DNA by a partial intercalation. Based on the above experiments, the results have clearly indicated the different ability of the ligands in the three complexes [Co(phen)2(PIP)]3, 3 [Co(phen)2(HPIP)] and [Co(phen)2(HNAIP)]3, to stack and overlap with the base pairs. Although the crystal structure of these complexes are unavailable at present, in our previous studies, we have got the crystal structure of the ligand PIP, in which the phenyl ring is almost coplanar with the imidazole ring. Herein, we think that the ortho phenolic group of HPIP can also be well coplanar with the imidazole ring due to forming an intramolecular hydrogen-bond with the nitrogen atom of imidazole ring, which have also been seen with analogous compounds in their crystal structures [34 / 38]. The introduction of ortho phenolic group to the HPIP molecule may have some additional affinity to stabilize the complexes binding to DNA. While in the ligand HNAIP, the large naphthyl ring may lie out of the plane of the imidazole ring, decreasing the planarity of ligand, thus resulting in the partial intercalation of the HNAIP ligand. These results parallel to the above absorption spectra and emission spectra of both complexes in the presence of DNA. 3.5. Photocleavage Besides the above methods, the binding of the two complexes to DNA was also investigated by DNA photocleavage assay. Irradiation of complexes [Co(phen)2(HPIP)]3 or [Co(phen)2(HNAIP)]3 and plasmid in tris /acetate buffer (pH 7.2) at 302 nm under aerobic conditions results in the cleavage of supercoiled form I of the plasmid pBR 322 DNA to the nicked form II (Fig. 4). No DNA cleavage was observed for controls in which the complex was absent (Fig. 4, lane 0), or incubation of the plasmid with either complex in dark
(data not presented). As increasing the concentration of [Co(phen)2(HPIP)]3 and [Co(phen)2(HNAIP)]3, the amount of form II increases gradually, while form I diminishes gradually. On the other hand, the complexes exhibited different cleaving efficiency for the plasmid DNA. [Co(phen)2(HPIP)]3 can induce the obvious cleavage of the plasmid DNA at the concentration of 20 mM. However, [Co(phen)2(HNAIP)]3 exhibited much lower cleaving efficiency for DNA. Even at the concentration of 80 mM, [Co(phen)2(HNAIP)]3 cannot promote the complete conversion of DNA from form I to II. The different DNA-cleavage efficiency of the two complexes was due to the different binding affinity of the complexes to DNA. In order to establish the reactive species such as singlet oxygen, superoxide anion radical and hydroxyl radicals that are responsible for the photoactivated cleavage of the plasmid DNA, the following experiments are carried out. For [Co(phen)2(HPIP)]3, firstly, the cleavage was performed in the presence of D2O to test the possibility that the photoactivated cleavage involves the formation of singlet oxygen, which is known to react with guanine residues at neutral pH. Singlet oxygen would be expected to induce more strand scissions in D2O than in H2O, owing to its longer lifetime in the former solvent [41]. As seen in Fig. 5(A), lane 3, no enhancement but inhibition was observed. This indicated that the singlet oxygen was not the active species in the cleavage reaction. Furthermore, studies with the singlet oxygen quencher histidine were carried out [42], and no obvious inhibition was observed (Fig. 5(A), lane 2), which further confirmed that singlet oxygen was not involved in the cleavage. On the other hand, in the + presence of a superoxide anion radical (O2 ) scavenger such as superoxide dismutase (SOD) (1000 u ml1) (Fig. 5(A), lane 4), the cleavage was obviously inhibited, indicating that superoxide anion radical is likely the reactive species. At the same time, in the presence of different hydroxyl radical scavengers such as mannitol [43] (50.0 mM) (Fig. 5(A), lane 5), ethanol (1.7 mM) (Fig. 5(A), lane 6), sodium formate (100.0 mM) (Fig. 5(A), lane 7), and DMSO (10.0 mM) (Fig. 5(A), lane 8), the different degrees of inhibition in the photoinduced
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properties were also studied. Spectroscopic studies together with cyclic voltammetry and viscosity experiments supported that [Co(phen)2(HPIP)]3 bound to DNA by intercalation, and exhibited stronger binding affinity to DNA. While [Co(phen)2(HNAIP)]3 bound to DNA by a partial intercalative mode, and exhibited relatively lower binding affinity. In addition, both complexes have been found to promote the singlestranded cleavage of plasmid pBR 322 DNA upon irradiation. The cleaving mechanisms between complexes and DNA were also proposed.
Acknowledgements
Fig. 5. Photoactivated cleavage of pBR 322 (0.1 mg) in the presence of complex and different inhibitors after irradiation at 302 nm for 60 min. (A) In the presence of 40 mM [Co(phen)2(HPIP)]3 : (0) no complex; (1) no inhibitor; (2) in histidine (1.2 mM); (3) D2O used instead of buffer; (4) in SOD (1000 u ml 1); (5) in mannitol (50.0 mM); (6) in ethanol (1.7 mM); (7) in sodium formate (100.0 mM); (8) in DMSO (10.0 mM). (B) In the presence of 40 mM [Co(phen)2(HNAIP)]3 : (0) no complex; (1) no inhibitor; (2) in histidine (1.2 mM); (3) D2O used instead of buffer; (4) in SOD (1000 u ml 1); (5) in mannitol (50.0 mM); (6) in ethanol (1.7 mM); (7) in sodium formate (100.0 mM); (8) in DMSO (10.0 mM).
cleavage of the plasmid by [Co(phen)2(HPIP)]3 were also observed, suggesting that in addition to the superoxide anion radical, hydroxyl radical is also likely to be one of the reactive species for the cleavage. For the complex [Co(phen)2(HNAIP)]3, the same experiments were also conducted to investigate the reactive species responsible for the cleavage reaction. From Fig. 5(B), it can be seen that in the presence of histidine, no obvious inhibition was observed. Furthermore, no obvious enhancement was observed in D2O (lane 3), this indicated that the singlet oxygen was not the reactive specie in the cleavage. In addition, in the presence of the SOD, the cleavage was also not inhibited, indicating that superoxide anion radical + (O2 ) was not involved in the cleavage. However, in the presence of different hydroxyl radical scavengers such as mannitol (lane 5), ethanol (lane 6), sodium formate (lane 7), and DMSO (lane 8), the different degrees of inhibition in the photoinduced cleavage of the plasmid were observed, this suggested that the hydroxyl radical is likely to be the reactive species for the cleavage reaction.
4. Conclusions Two complexes [Co(phen)2(HPIP)]3 and 3 [Co(phen)2(HNAIP)] have been synthesized and characterized. Their DNA-binding and photocleavage
We are grateful to the National Natural Science Foundation of China, the Natural Science Foundation of Guangdong Province, the State Key Laboratory of Bio-organic and Natural Products Chemistry in Shanghai Institute of Organic Chemistry, the State Key Laboratory of Coordination Chemistry in Nanjing University, and the Visiting Scholar Foundation of Key Laboratory in University for their financial support.
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