DNA cleavage by hydroxy-salicylidene-ethylendiamine-iron complexes (original) (raw)

Abstract

Bis(hydroxy)salen Fe complexes were designed as self-activated chemical nucleases. The presence of a hy-droxyl group on the two salicylidene moieties serve to form a hydroquinone system cooperating with the iron redox system to facilitate spontaneous formation of free radicals. We compared the DNA binding and cleaving properties of the ortho-, mete- and para-(bishydroxy) salen Fe complexes with that of the corresponding chelate lacking the hydroxyl groups. DNA melting temperature studies indicated that the para complex exhibits the highest affinity for DNA. In addition, this para compound was considerably more potent at cleaving supercoiled plasmid DNA than the regio-isomeric ortho- and meta-hydroxy-salen·Fe complexes, even in the absence of a reducing agent, such as dithiothreitol used to activate the metal complex. The DNA cleaving activity of the para isomer is both time and concentration dependent and the complexed iron atom is absolutely essential for the sequence uniform cleavage of DNA. From a mechanistic point of view, electron spin resonance measurements suggest that DNA contributes positively to the activation of the semi-quinone system and the production of ligand radical species responsible for subsequent strand scission in the absence of a reducing agent. The para-hydroxy-salen-Fe complex has been used for detecting sequence-specific drug-DNA interactions. Specific binding of Hoechst 33258 to AT sequences and chromomycin to GC sequences were shown. The para -bis(hydroxy)salenFe derivative complements the tool box of footprinting reagents which can be utilised to produce efficient cleavage of DNA.

Introduction

Salicylidene-ethylendiamines, commonly known as salens, can form stable complexes with a variety of transition metals.

These metal Schiff base complexes have been developed as catalysts for the epoxidation of olefins ( 1 ) and as multidimensional magnetic compounds ( 2 ). In addition, salen-metal complexes have been designed as nucleic acids reagents to induce specific damages in DNA or RNA. For example, salen-Mn III complexes were shown to produce enantiospecific recognition and cleavage of right-handed double helical DNA ( 3 ). Salen-Ni II complexes were used for the cleavage of guanine structures in DNA and RNA ( 4 ). The macrocyclic salen-type chelate Ni·TMAPES is a useful tool in molecular biology to probe the structure of RNA via selective alkylation of unpaired guanine residues ( 5 ). DNA cleavage can also be induced by salen-Cu II complexes. In a previous study, we showed that a functionalised salen·Cu chelate binds in the groove of the double helix and efficiently cleaves DNA in the presence of a reducing agent ( 6 ). This copper complex was subsequently linked to a variety of DNA-binding ligands, including both intercalators and minor groove binders, to produce salen-based chemical nucleases, some of which exhibited marked cytotoxic activities against tumour cells ( 7 , 8 ). Salen-Co II complexes ( 9 ) and, recently, salen-Ru III complexes were also designed as DNA cleavers ( 10 ).

Mn III ·salen and Ni II ·salen complexes mediate the cleavage of DNA in the presence of terminal oxidants. In contrast, due to differing redox properties, a reducing cofactor is required to initiate the cleavage reaction of DNA with Cu II ·salen and Co II ·salen complexes. Mercaptopropionic acid provides an efficient activator for salen·Cu II complexes ( 6 , 8 ). But in a very recent study, we showed that efficient cleavage of DNA can be achieved with a new type of salen·Cu complex in the absence of any activating agent. With the design of bis(hydroxy)salen·Cu complexes, we were able to create a hydroquinone system cooperating with the copper redox system to facilitate the spontaneous formation of oxidising Cu III species involved in the cleavage of DNA ( 11 ).

Our recent study ( 11 ) revealed that the redox properties and therefore the DNA cleaving activities of bis(hydroxy)-salens·Cu complexes depend crucially on the position of the hydroxyl groups. To investigate further the role of these substituents, we decided to synthesise a novel series of salen·Fe complexes bearing hydroxyl groups in ortho, in meta or in para on the aromatic ring of the salen moiety ( Fig. 1 ). The DNA cleaving properties of the new complexes are reported here. This is the first example of salen·Fe complexes used as nucleases.

Materials and Methods

Chemistry

Hydroxysalen·metal complexes were obtained using a conventional procedure for salen synthesis based on the condensation of hydroxysalicylaldehyde with ethylenediamine, as previously described for salen·Cu complexes ( 6 , 11 ). The iron complex formation was achieved in the presence of ferrous chloride. Briefly, FeCl 2 tetrahydrate (0.298 g, 0.170 mmol) was added under Argon to bis(hydroxy)salen (0.5 g, 0.166 mmol) ( ortho, meta or para ) in 30 ml absolute ethanol and 5 ml dry acetone. The solid suspension was refluxed under vigorous stirring for 1 h. The mixture was then cooled to room temperature and oxidised for 10 min with air bubble. The resulting suspension was filtered, washed with methanol (2 = 10 ml) and dried with ethyl ether (2 = 10 ml) to afford the desired iron complexes. 3a ortho: yield 82% (0.531 g); black solid; m.p. > 230°C; IR (KBr, cm -1 ) v 3400, 3000, 1640, 1600, 1540, 1480, 1450, 1250, 1220, 1080, 1040, 1020, 860, 790, 750; MS (MALDI+) 354.2 M+. 3b meta: yield 77% (0.500 g); black solid; m.p. > 230°C; IR (KBr, cm -1 ) v 3200, 1600, 1540, 1500, 1390, 1340, 1220, 1180, 1140, 1030, 1010, 990, 850, 810, 640; MS (MALDI+) 354.0 M+. 3c para: yield 90% (0.579 g); black solid; m.p. > 230°C; IR (KBr, cm -1 ) v 3400, 1620, 1600, 1560, 1540, 1470, 1440, 1380, 1320, 1280, 1250, 1220, 1160, 1020, 880, 820, 800; MS (MALDI+) 435.3 (M + C1 + EtOH)+.

The synthesis of the non-hydroxylated salen (metal-free 1) has been reported ( 6 ). A similar procedure was used to synthesise the amino compound (metal-free 2). Briefly, NaBH 4 (0.141 g) was added in small portions to a solution of salen compound (0.5 g) in 20 ml dry methanol at room temperature. After 1 h stirring, 10 ml of cold water were added and the salen compound was extracted with CH 2 C1 2 (2 = 50 ml). The organic layer was washed with water (2 = 50 ml). After drying over Na 2 SO 4 the solvent was removed by distillation under reduced pressure. The amino compound was obtained as a white-orange solid (0.484 g; 90%); m.p. 120°C, Rf (MeOH/CHCl 3 10:90) 0.13; 1 H-NMR (DMS0-D 6 ) δ 2.83 (s, 4H), 3.98 (s, 4H), 6.78 (t, J. = 7.4 Hz, 2H), 6.82 (t, J. = 8.1 Hz, 2H), 6.97 (d, J. = 7.3 Hz, 2H), 7.17 (t, J = 7.4 Hz, 2H); 13 C-NMR (DMS0-D 6 ) δ 47.70, 52.55, 116.56, 119.24, 122.28, 128.53, 128.88, 157.99. Complex formation was achieved as described above for the bis(hydroxy)salen analogues.

Chemicals and biochemicals

Hoechst 33258 was purchased from Sigma Chemical Co. A sample of bisantrene was kindly supplied by Dr Manlio Palumbo of University of Padova, Italy. Calf thymus DNA and the double-stranded polymer poly(dA-dT)-poly(dA-dT) were from Pharmacia (Uppsala, Sweden). Calf thymus DNA was deproteinised with sodium dodecyl sulphate (protein content <0.2%). [α- 32 P]dATP (3000 Ci/mmol) was purchased from Amersham (Buckinghamshire, UK). Restriction endonucleases Aval, Eco RI and Pvu II, and AMV reverse transcriptase were purchased from Boehringer (Mannheim, Germany) and used according to the supplier's recommended protocol in the activity buffer provided. The plasmids pBS (Stratagene, La Jolla, CA) and pAT were prepared from Escherichia coli according to standard procedures employing sodium dodecyl sulphate-sodium hydroxide lysis followed by purification using Qiagen columns.

Melting temperature studies

Melting curves were measured using a Uvikon 943 spectro-photometer coupled to a Neslab RTE111 cryostat. For each series of measurements, 12 samples were placed in a thermostatically controlled cell-holder, and the quartz cuvettes (10 mm pathlength) were heated by circulating water. The measurements were performed in BPE buffer pH 7.1 (6 mM Na 2 HPO 4 , 2 mM NaH 2 PO 4 , 1 mM EDTA). The temperature inside the cuvette was measured with a platinum probe; it was increased over the range 20–100°C with a heating rate of 1°C/ min. The ‘melting’ temperature _T_m was taken as the mid-point of the hyperchromic transition.

DNA purification and labelling

The 117 and 265 bp DNA fragments were prepared by 3′- 32 P-end labelling of the EcoR I -Pvu II double digest of the pBS plasmid using [α- 32 P]dATP and AMV reverse transcriptase. Similarly, the 160mer tyri T fragment was prepared by 3′-end labelling of the EcoR I -Ava I digest of plasmid pAT ( 12 ). In each case, the digestion products were separated on a 6% polyacrylamide gel under native conditions in TBE buffered solution (89 mM Tris-borate pH 8.3, 1 mM EDTA). After autoradiography, the band of DNA was excised, crushed and soaked in water overnight at 37°C. This suspension was filtered through a Millipore 0.22 μ filter and the DNA was precipitated with ethanol. Following washing with 70% ethanol and vacuum drying of the precipitate, the labelled DNA was resuspended in 10 mM Tris, adjusted to pH 7.0, containing 10 mM NaCl.

DNA cleaving activity

Experiments with plasmid DNA on agarose gels . Each reaction mixture contained 4 μl of supercoiled pAT DNA (0.6 μg in a Tris-HCl, 10 mM NaCl buffer at pH 7.0), 4 μl of water, 10 μl of the salen compound at the desired concentration and in some cases 2 μl of 1 mM dithiothreitol. After incubation at 37°C for the indicated time, 4 μl of loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol in H 2 O) were added to each tube and the solution was loaded onto a 1% agarose gel. The electrophoresis was carried out for ∼2 h at 100 V in TBE buffer (89 mM Tris-borate pH 8.3, 1 mM EDTA). Gels were stained with ethidium bromide (1 (Ag/ml) then destained in water prior to being photographed under UV light.

Experiments with 32 P-labelled DNA on sequencing gels . The DNA restriction fragment, 117 or 160 bp in length, was treated with the metal complex in the presence of 200 μM dithiothreitol, in a volume of 20 μl, for 30 min at room temperature and then precipitated with cold ethanol. The DNA pellet was resuspended in 5 μl of 80% formamide containing TBE buffer and 0.1% tracking dyes. Samples were heated to 90°C for 4 min and then chilled in an ice bath just before being loaded on a sequencing gel (8% polyacrylamide, 7 M urea). For the footprinting experiments, the labelled DNA (6 μl, ∼500 c.p.s.) was mixed with 10 μl of Hoechst 33258, doxorubicin, chromomycin or bisantrene at the desired concentration and the drug-DNA complex was left to equilibrate for at least 30 min at 37°C.

Figure 1

Structure of salen-Fe complexes.

Successively, 2 μl of salen-Fe (100 μM) and 2 μl of a freshly prepared 1 mM solution of dithiothreitol were added to initiate the cleavage reaction. After 30 min incubation at 37°C, the reaction was stopped by adding cold ethanol to precipitate the DNA. After centrifugation and two washes with 70% ethanol, DNA in each tube was resuspended in 5 μl of formamide-TBE loading buffer.

Electron spin resonance (ESR)

X-band ESR spectra were obtained using a Bruker ESC 106 or a Varian E-9 operating at 100 kHz modulation frequency at room temperature (20°C) or at liquid nitrogen temperature (77K). The g factor measurements were related to the ‘strong pitch’, g = 2.0028. For spin-trapping experiments 5 mM freshly distilled 5,5′-dimethyl pyrroline- N -oxide (DMPO; Sigma Chemical Co.) was added to the solutions of the salen-Fe complex at 1 mM in DMSO or methanol/water (1:1) mixture.

Results

The DNA binding and cleaving properties of the ortho -( 3a ), meta -( 3b ) and para-(bishydroxy)salen-Fe complexes ( 3c ) were compared with that of the corresponding chelate lacking the hydroxyl groups (compound 1 in Fig. 1 ). We also used the amino compound 2 as a control.

Thermal denaturation

To evaluate the propensity of the metal complexes to bind to DNA, we measured their ability to alter the thermal denaturation profile of DNA. The change of the absorbance at 260 nm was recorded as a function of the temperature for calf thymus DNA and poly(dA-dT)·(dA-dT). It was not possible to study the effects on poly(dG-dC)·(dG-dC) due to its high stability even at very low ionic strength. The variation of the _T_m (ΔT m ) of helix-to-coil transition of calf thymus DNA and of the polynucleotide (20 μM each) was determined in the presence of 10 μM ligand. The Δ _T_m values, which correspond to the difference between the _T_m measured in the presence of the ligand and that obtained with the free DNA, are given in the histogram in Figure 2 .

Figure 2

Variation in difference melting temperature (Δ _T_m = _T_mligand-DNA - _T_mDNA ) measured with the salen-Fe complexes interacting with calf thymus DNA or the polynucleotide poly(dA-dT)·poly(dA-dT). _T_m measurements were performed in BPE buffer pH 7.1 (6 mM Na 2 HPO 4 , 2 mM NaH 2 PO 4 , 1 mM EDTA) using 20 μM DNA and 10 μM metal complex, in 3 ml quartz cuvettes at 260 nm with a heating rate of 1 °C/min. Each measurement was performed in duplicate. The _T_m of calf thymus DNA and poly(dA-dT)·poly(dA-dT) alone are 66 and 41°C (±1°C), respectively.

A large increase in the _T_m value of nucleic acids is observed for the para isomer bound to CT-DNA or polynucleotides. The stabilisation of the DNA double helix by binding of the ortho and meta salen·Fe complexes is considerably weaker. Very little effect was observed with the non-hydroxylated iron complexes 1 and 2. With calf thymus DNA, which contains roughly equal proportions of A·T and G·C base pairs, the stabilising action of the regio-isomers ranks in the order: para >> ortho > meta . The para complex 3c exhibits the highest affinity for DNA.

DNA cleavage

Iron complexes such as EDTA·Fe generally act as potent DNA cleavers in the presence of a reducing agent which serves to maintain the ferrous form of the metal. For this reason, we compared the DNA cleaving activity of the different salen·Fe complexes in the absence and presence of a reducing agent, dithiothreitol. Supercoiled plasmid DNA was incubated with the salen·Fe complexes (50 μM each), with or without 200 μM dithiothreitol, for 3.5 h at 37°C. As shown in Figure 3A , practically no cleavage occurs with the amino compound 2 and only little cleavage was detected with salen 1 lacking the hydroxyl groups. With both 3a and 3b, the DNA cleaving activity is very modest since only 6 ± 2% of the supercoiled DNA (form I) was cleaved to generate the nicked DNA (form II) after 3.5 h of incubation with the salen. Interestingly, 3c was considerably more potent at cleaving DNA. In the absence of the reducing agent, ∼75 + 5% of the supercoiled DNA was converted to nicked DNA within the 3.5 h period of incubation at 37°C. Dithiothreitol further activates the reaction since in this case the plasmid DNA was totally converted into the nicked form II. The same types of gels were obtained when we varied the time of the reaction. A typical gel obtained after a long incubation period of the circular DNA with the salen complexes for 20 h is presented in Figure 3B . Here again, it can be seen most clearly that the para complex is much more potent than the two other regio-isomers. The band of nicked DNA represents ∼18 ± 7% with compounds 1 and 2 as well as with 3a and 3b . With these complexes, the addition of dithiothreitol has no significant effect on the DNA cleavage activity which always remains relatively weak, even after such a long incubation period. In contrast, complex 3c cleaves DNA very efficiently, even in the absence of the reducing agent. After 20 h incubation without dithiothreitol, there was no more supercoiled DNA and a faint band of form III DNA can be detected. This band corresponding to linear DNA is much stronger when the cleavage reaction was carried out in the presence of 200 μM dithiothreitol. Now about half of the initial DNA content was found in the nicked form II and the other half was converted to linear DNA.

Figure 3

Cleavage of closed circular DNA. Supercoiled DNA (0.6 μg) was incubated at 37°C for 3.5 h ( A ) or for 20 h ( B ), with the salen·iron complexes at 50 μM, in the absence (-) or presence (+) of 200 μM dithiothreitol. Forms I, II and III refer to the supercoiled, nicked and linear DNA forms, respectively. The DNA samples were resolved on a 1% agarose gel. Control tracks labelled ‘Ct’ contained no ligand.

We studied the DNA cutting activity of the para-hydroxy-salen·Fe complex at different concentrations. Figure 4 shows the concentration-dependent cleavage of DNA by 3c in the presence of dithiothreitol. The reaction was carried out for 3 ( Fig. 4A ) or 8 h ( Fig. 4B ) at 37°C. In both cases, the supercoiled DNA is gradually converted to nicked DNA. It is important to note that the same para -hydroxy-salen compound not complexed to Fe has absolutely no cleavage activity. The metal is absolutely required for the cutting reaction to proceed.

Next, we investigated the cleavage activity of the para complex 3c using DNA restriction fragments, 117 and 160 bp in length. The 3′-end radiolabelled DNA samples were incubated with increasing concentrations of the salen·Fe complex, in the presence of the reducing agent. After 30 min incubation, the samples were precipitated and the integrity of the DNA was analyzed by electrophoresis on acrylamide gel under denaturing conditions. The cleavage reaction is essentially non-specific ( Fig. 5 ). With 25 μM salen-Fe the cleavage is not very efficient but at 50 and 100 μM the DNA is cleaved almost randomly. Cleavage of the DNA fragment by the para -hydroxy-salen-Fe complex affords an almost uniform ladder of bands. However, homopolymeric AT stretches are cleaved less efficiently than alternating sequences. The patterns are reminiscent of those obtained with other iron complexes, such as EDTA·Fe II and MPE·Fe II , and suggest that diffusible radicals are responsible for the non-specific cleavage of DNA.

Figure 4

Concentration dependence for the cleavage of plasmid DNA by 3c . Supercoiled DNA (0.6 μg) was incubated at 37°C for 3 h ( A ) or for 8 h ( B ) with the iron complex, in the presence of 200 μM dithiothreitol. The concentration (μM) of the metal complex is shown at the top of the appropriate gel lanes. Lanes marked p-salen refer to the iron-free para -hydroxy-salen. Other details as for Figure 3 .

Use as a probe for detecting sequence-selective drug-DNA interactions

Because the cutting is not sequence selective, as is the case with most chemical nucleases ( 13 ), we reasoned that the salen complex may be used as a footprinting probe to investigate binding of proteins and drugs to specific sequences in DNA. This hypothesis was tested experimentally. The results of foot-printing experiments are presented in Figure 6 . The 160 bp tyf T DNA fragment was incubated with either the benzimidazole drug Hoechst 33258 or the anthracene derivative bisantrene. Hoechst 33258 is a well known DNA minor groove binder which binds preferentially to AT-rich sequences in double-stranded DNA ( 14 ). Bisantrene is an antitumour agent which intercalates into DNA, with little sequence preference ( 15 ). The DNA bound to Hoechst 33258 or bisantrene was subjected to limited cleavage with 3c for 30 min and the DNA cleavage products were resolved on a sequencing gel. In the control lane, incubated without DNA-binding drug, the cleavage of DNA is totally uniform, as observed in Figure 5 . When the DNA was incubated with the minor groove binder Hoechst 33258, two areas of decreased cleavage intensity can be detected around nucleotide positions 30 and 48. They both correspond to sequences containing at least six contiguous A·T bp. Another AT-rich site can be discerned near the top of the gel around position 85 but it lies beyond the region accessible for analysis by phosphorimaging. Such AT tracts are known to provide high affinity binding sites for Hoechst 33258 ( 16 ). Selective binding to AT-rich sequences was also detected using the antibiotic netropsin (data not shown). The cleavage of DNA is reduced in the presence of bisantrene but no specific binding site can be detected. Previous footprinting experiments with the enzyme DNase I have revealed a weak preference for alternating purine-pyrimidine sequences ( 15 ). This preference is too weak to be detected using chemical footprinting probes, including the salen-Fe complex 3c.

Figure 5

Non-specific cleavage of the 117 bp DNA fragment by 3c . The DNA was 3′-end labelled with [α- 32 P]dATP and AMV reverse transcriptase. The cleavage products were resolved on an 8% polyacrylamide gel containing 8 M urea. The concentration (μM) of the p -salen complex is shown at the top of the appropriate gel lanes. G, DMSO markers specific for guanines. Ct, DNA alone, incubated without ligand; this sample serves as a control to assess background nicking of the DNA preparation. Numbers on the right side of the gel refer to the numbering scheme of the DNA fragment.

Figure 6

Salen-Fe footprinting of Hoechst 33258 and bisantrene (Bst) on a 160 bp Ava I -Eco RI restriction fragment cut out from plasmid pAT. The control lane (Ct) shows the products resulting from limited digestion of DNA by 3c in the absence of drugs. The lane marked ‘DNA’ contains the 32 P-labelled DNA alone, incubated without drug or salen-Fe. The remaining lanes show the products of salen·Fe digestion in the presence of 25 and 50 μM drug. The sequences of the two Hoechst 33258 binding sites protected from cleavage by the nuclease are indicated. Other details as for Figure 5 .

Similarly we could not detect clear footprints with another classical intercalating agent, doxorubicin. Previous footprinting studies with DNase I have revealed that this anticancer drug interacts preferentially with 5′-(A/T)CG and 5′-(A/T)GC triplets ( 17 ) but the sequence selectivity is too weak to be detected with our salen·Fe probe. However, the drug stimulates the cleavage of DNA by the metal complex at several sites (marked with an asterisk in Fig. 7 ) which essentially correspond to AT-rich sequences. Sequences containing consecutive A-T bp are known not to provide good binding sites for doxorubicin ( 17 , 18 ). In contrast, we could easily detect the sequence-selective binding of chromomycin to GC-rich sequences in DNA. Chromo-mycin is a minor groove binding agent which exhibits a sharp preference for GC-rich sequences ( 19 , 20 ). The footprinting gel in Figure 7 reveals unambiguously that the drug recognises a number of sites within the 265 bp target fragment. The footprints (marked with open rectangles) coincide with GC-rich sites and are flanked with sequences (mostly AT-rich) where the cleavage by the chemical probe 3c has been significantly enhanced (marked with asterisks along the gel lane). The chemical nuclease 3c is most efficient at detecting sequence-selective interactions between minor groove binders and AT-or GC-rich sequences in DNA.

Figure 7

Salen-Fe footprinting of doxorubicin (Doxo.) and chromomycin on a 265 bp Pvu II -Eco RI restriction fragment cut out from plasmid pBS. The lane marked ‘DNA’ contains the 32 P-labelled DNA alone, incubated without drug or salen-Fe. The control lane (Ct) shows the products resulting from limited digestion of DNA by 3c in the absence of drugs. The lane marked ‘DTP’ contains the radiolabelled DNA incubated with dithiothreitol but without salen-Fe. The other lanes show the products of salen-Fe digestion in the presence of the test drug at the indicated concentration (μM). The sequences of the chromomycin binding sites protected from cleavage by the nuclease are indicated by open rectangles and the regions of enhanced cutting are marked with asterisks. Other details as for Figure 5 .

Mechanism of DNA cleavage

The ESR spectrum of a 1 mM methanolic solution of 3c in the absence of DNA is presented in Figure 8A . The spectrum with g⊤ = 6.07 and g ≈ 2, obtained in liquid nitrogen (77 K), is characteristic of a high spin Fe III complex with axial geometry. In the presence of DNA ( Fig. 8C ), the intensity of the ESR signal at g⊤ = 6.07 decreases significantly (>50%) and the line is doubled with a broadening effect. This suggests that a radical, formed in the presence of DNA, interacts with a paramagnetic center. The drop of signal intensity can be attributed neither to a formation of higher iron oxidation state species nor to lower oxidation state species. Indeed, a similar doubling and broadening effect of the signal at g ⊤ = 6.07 was obtained after addition of a small amount of NaOH in order to activate the hydro-quinonic system ( Fig. 8B ). The same double signal at g ⊤ = 6.07 was observed in addition to the specific semi-quinone radical at a g value of ∼2. But in this case, the signal intensity is not significantly affected. Therefore, it is most likely that the decreased intensity of the ESR signal observed in the presence of DNA is not due to an antiferromagnetic process resulting from two radical spin interaction. DNA may contribute to the activation of the semi-quinone system and the subsequent production of ligand radical species responsible for DNA cleavage in the absence of a reducing agent. The quinone system likely cooperates with the iron moiety to facilitate the formation of salen·Fe(III)·O 2 - species or similar higher oxidation state activated salen species (e.g. Fe IV,V = O) and then ultimately to produce free radicals responsible for DNA cleavage. A similar mechanism is known for the antitumour drug bleomycin ( 21 ).

Figure 8

ESR spectra of a 1 mM methanolic solution of 3c in the absence ( A ) or presence of 1 mM sodium hydroxyde ( B ) or 300 μM calf thymus DNA ( C ). Experimental settings: 10 G modulation amplitude, 2000 G field sweep and scan rate 4 min. The receiver gain was 10 3 for (A) and (B), and 5 × 10 3 for (C).

The situation is simpler in the presence of a reducing agent. With added dithiothreitol, reactive oxygen species were detected by spin-trapping experiments performed using DMPO as the spin-trapping agent. When a water/methanol mixture was used as a solvent, only a weak signal of super-oxide anion was detected (data not shown). But with DMSO as a solvent, a strong signal of DMPO-OH spin adduct with hyperfme splittings constant a N = a H = 14.5 G was detected ( Fig. 9 ). Such an adduct typically results from the decomposition of superoxide anion in DMSO and the production of hydoxyl radical ( 22 ). By analogy with the mechanism of DNA cleavage of bleomycin ( 21 ), it is likely that the formation of free radicals such as ·OH and O 2 - results from the heterolytic cleavage of DNA-bound salen·Fe III -OOH species.

Figure 9

ESR spectrum of the DMPO-OH radical adduct (a N = a H = 15.2 G) obtained with complex 3c at room temperature. Experimental settings: 20 mW power, 1 G modulation amplitude, gain 3.2 × 10 4 and scan rate 4 min.

Discussion

In recent years, we have developed series of salen·metal complexes containing Ni, Co, Mn or Cu ( 6–8 , 23 ). In most cases, but not all, these metal complexes revealed DNA cleavage activities but the cutting efficiency was generally weak compared to what can be achieved with conventional metal complexes such as EDTA·Fe. For example, none of our previous salen-metal complexes was sufficiently potent to be used a footprinting probe. Recently, we decided to incorporate hydroxyl groups on the salen structure, not only to increase the solubility of the complexes in aqueous solution but also to promote the DNA cutting activity. In a recent publication, we reported that the incorporation of hydroxyl groups on salen-Cu complexes can reinforce the stability of the copper complex and creates a hydroquinone system cooperating with the copper redox system to facilitate the spontaneous formation of oxidising Cu m species ( 11 ). We showed that the DNA cleaving activities of the salen-Cu complexes depend crucially on the position of the OH groups. In the copper series, the para isomer cleaved DNA much more efficiently that the ortho isomer and the meta isomer was inactive ( 11 ). A good correlation between the nuclease activity of the hydroxy-salen·Cu complexes and their ability to form the oxidising Cu m species was demonstrated.

The situation reported here with the hydroxy-salen·Fe complexes is similar. Complex 3c is by far the most efficient DNA cleaver in the series. Both the OH groups in the para position and the iron metal are essential to the DNA cleavage activity ( Fig. 4 ). By analogy with our previous study with the copper complexes, we can propose that the hydroxyl groups in the para position act as a quinone system which would cooperate with the iron moiety to facilitate the formation of free radicals responsible for DNA cleavage. This hypothesis is supported by the ESR data. An important detail revealed by this study is that DNA contributes significantly to the activation of the nuclease 3c. The para complex exhibits a considerably higher affinity for DNA than the ortho and meta analogues. The interaction of the para complex with DNA clearly serves to facilitate the activation of the radical system, as revealed by the ESR spectrum presented in Figure 8C . In other words, DNA (the victim) greatly helps to induce its own degradation by the para- hydroxy-salen·Fe complex (the killer). As shown here, salen·Fe complexes such as 3c may be used as footprinting probes to investigate sequence-specific drug-DNA interactions. This probe, which is a rare example of a self-activated chemical nuclease, may also prove useful for detecting preferential binding of proteins and peptides to specific sequences in DNA.

Acknowledgments

C.B. thanks Brigitte Baldeyrou and Nathalie Gelus for expert technical assistance with the DNA cleavage and footprinting experiments.

References

1

,

Chem, Soc. Rev.

,

1999

, vol.

28

(pg.

85

-

93

)

2

,

Angew. Chem,. Int. Ed. Engl

,

1995

, vol.

34

(pg.

1446

-

1448

)

3

,

Inorg. Chem.

,

1996

, vol.

35

(pg.

4837

-

4847

)

4

,

Acc. Chem. Res.

,

1994

, vol.

27

(pg.

295

-

301

)

5

,

Nucleic Acids Res.

,

1993

, vol.

21

(pg.

5524

-

5525

)

6

,

J. Org. Chem.

,

1996

, vol.

61

(pg.

2326

-

2331

)

7

,

Bioorg. Med. Chem.

,

1996

, vol.

4

(pg.

1185

-

1196

)

8

,

Bioorg. Med. Chem. Lett.

,

1997

, vol.

7

(pg.

63

-

66

)

9

,

Bioconjugate Chem.

,

1997

, vol.

8

(pg.

798

-

812

)

10

,

J. Chinese Chem. Soc

,

1998

, vol.

45

(pg.

611

-

617

)

11

,

J. Am. Chem. Soc

,

1999

, vol.

121

(pg.

1862

-

1869

)

12

,

EMBO J.

,

1985

, vol.

4

(pg.

1025

-

1032

)

13

,

Chem. Rev.

,

1993

, vol.

93

(pg.

2295

-

2316

)

14

,

J. Mol. Biol.

,

1997

, vol.

271

(pg.

244

-

257

)

15

,

Mol. Pharmacol.

,

1998

, vol.

54

(pg.

1036

-

1045

)

16

,

Biochim. Biophys. Acta

,

1987

, vol.

949

(pg.

158

-

168

)

17

,

Biochemistry

,

1990

, vol.

29

(pg.

6145

-

6153

)

18

,

Biochemistry

,

1998

, vol.

37

(pg.

1033

-

1045

)

19

,

Biochemistry

,

1983

, vol.

22

(pg.

2373

-

2377

)

20

,

Biochemistry

,

1992

, vol.

31

(pg.

9310

-

9318

)

21

,

Chem. Rev.

,

1998

, vol.

98

(pg.

1153

-

1169

)

22

,

J. Biol. Chem.

,

1986

, vol.

261

(pg.

4426

-

4431

)

23

,

Bioorg. Med. Chem. Lett.

,

1997

, vol.

7

(pg.

63

-

66

)

Author notes

Present address: Sylvain Routier, ICOA, UPRESA 6005, Université d'Orléans, 45067 Orléans, France

© 1999 Oxford University Press