Development of Novel DNA Cleavage Systems Based on Copper Complexes. Synthesis and Characterisation of Cu(II) Complexes of Hydroxyflavones

Copper(II) complexes of several hydroxyflavones were prepared and characterised through their physico-chemical properties. The nuclease activity of three synthesised complexes is reported. These copper(II) complexes present more nuclease activity than the ligands and the copper(II) ion.


INTRODUCTION
The flavonoids are a group of natural substances reported as being present in all higher plants. The basic flavone structure consists of 1,4-benzopyrone with phenyl substitution in position 2. The different groups of flavonoids differ in the number and position of substitutents on the aromatic ring, and in the extent and character of the oxidation on the pyrone portion of the molecule. Through their hydroxyl groups, the flavonoids are able to combine with sugars to form glycosides. In this sense, over 4000 different species of flavonoids have been described in fruits and vegetables, seeds, stems and flowers. They are therefore important component of the human diet. On average, a mixed Western diet supplies about g of flavonoids daily. The flavonoids exhibit a broad range of biochemical and pharmacological effects, some of which suggesting that a number of compounds of this group may affect the different cell systems. The flavonoids are probably the most common and active antioxidants in the human diet. In this sense, they are active both in hydrophilic and lipophilic systems. The mechanism responsible for the antioxidant activity appears to be dual. On one hand, they act as free radical scavengers, and on the other hand they are able to chelate metal ions, thereby reducing metal induced peroxidation.
Today it is known that the excessive production of free radicals and lipid peroxidation may be implicated in the development of certain chronic diseases such as atherosclerosis and cancer.
The quercetin (3',4',5,7-tetrahydroxyflavonol) and other 3-hydroxyflavones are degraded to depside and carbon monoxide by the quercetinase, a dioxygenase produced by the Aspergillusflavus. Quercetinase is a Cu(II)-containing dioxygenase. A Cu(II) chelate of quercetin is the key intermediate in the enzymatic reaction. In order to determine if the mode of coordination of flavones may give information relevant to the enzyme-quercetine interaction, Speier et al 3"s prepared copper(I) and copper (If) binary and ternary complexes of 3-hydroxyflavone, where the flavone interacts with the Cu(II) through the Ohydrox and the Ocarbonyl atoms. The synthesis and physicochemical properties of M(II) (Co, Ni and Cu) complexes with 5,7dyhroxyflavone was reported. 9 '1 On the other hand, Hadi et al. TM have shown that quercetin and other flavonoids cause strand scission in DNA in the presence of Cu(II) and that his reaction is associated with transient reduction of Cu(II) to Cu(1) and the generation of active species. Of the several metal ion tested [Cu(II), Fe(II), Fe(IIl), Co(II), Mn(II), Ni(II) and Ca(II)] only Cu(II) and Fe(III) complemented quercetin in the DNA breakage reaction. They proposed that the formation of a ternary complex of quercetin, DNA and Cu(II)/Cu(l) is involved in the generation of the oxygen active species that are the cause of the strand of DNA. A similar reaction dependent of metal ions and involving oxygen radicals has been reported with 1,10-phenantroline. 3 DNA-directed sequence-specific reagents capable of controlled chemical cleavage may also serve as molecular biological tools, either in the sense of standard but artificial restriction endonucleases or for a larger scale genome mapping.
With this in mind, it is important to develop not only novel DNA sequence reading molecules, but also chemical cleavage systems which are easily amenable to chemical synthesis manipulation and have suitably biocompatibility (e.g. stability, cellular penetration, recycling). 4 Recently several papers about the nuclease activity of copper complexes were reported. 15.26 Until now no paper concerning the nuclease activity of the copper complexes of flavones has been reported.
In the present paper we describe the synthesis and characterisation of a number of copper complexes of flavones (their structure is summarised in Figure 1) and the nuclease activity of three copper-flavone complexes. The antioxidant activity of these flavones were recently described
UV-Vis data were obtained on a Shidmazu 2101-PC instrument, while the infrared spectra were recorded as KBr disk on a Mattson Satellite Ftir spectrometer. X-band EPR spectra were collected at room temperature on a Bruker ER D200 spectrometer.
Anal. for Cu(3H,F)2, Found C 66.2 H 3.6 Cu 11.3 Calc. for C30HI806Cu C 66. 9 To a 50 ml of a methanolic solution containing mmol of Cu(OCH3)2, mmol of Hz7,8DHF was added with continuous stirring. After an hour approximately it appears a brown solid which is filtered, washed with methanol and air-dried.
Anal for Cu(3HF)(NH3)(H20), found C 62.8,; Cleavage of pBR322 and pUCI by Cu(II)-7,8DHF complexes A typical reaction was carried out by mixing 16 t-tl of the Cu(II) complex 0.625 tM in buffer Tris-HCI pH 8, 2 tl of 0.25 g/ktl pBR322 or pUCI and 2 ktl of H202 500 tM. The resulting solution contains 0.5 M of the complex, 0.025 lag/ktl pBR322 or pUCI and 50 t,tM of H202. After allowing the sample to incubate at 25 C for a 30 min, 3/al of a quench buffer solution consisting of a 0.2% bromophenol blue, 24% glycerol and mM edta, was added. Then the solution was subjected to electrophoresis on a 1% agarose gel in lx TBE buffer (0.1M tris borate, 0.2 mM edta) at 80 V about 4 h: The gel was stained with 0.5 lag/ml ethidium bromide and photographed on a UV transilluminator with a Polaroid camera MP4 containing an Agfapan (negative/positive) film.

RESULTS AND DISCUSSION
The band assigned to v(O-H) of the H3HF disappears in the IR spectrum of the Cu(3HF)2 complex. In the IR spectra of the rest of the synthesised complexes the existence of water or methanol molecules does not permit suggestions about the band attributed to v (O-H).
A shift of 20 cm 1 is observed for the band attributed to v(C=O) in the IR spectra of the complexes with respect to the ligands.
The v(C-O-C) bands remain unchanged in the metal complexes of H3HF an H25,7DHF with respect to the IR spectra of the ligands. However a shift of these vibrations take place in the IR spectra of the metal complexes of H27,8DHF (1080 in the complexes and 1160 cm " in the ligand). This is probably due to the deprotonation and coordination of the hydroxo groups at C7 and C8, neighbour of the C9-0-C 10 group.
According to the IR spectra it is possible to conclude that the 3HF" interacts with the Cu (II) ion as a bidentate ligand through the Ocarbony and Ohydroxo atoms in similarly as for the copper flavonolate complex. Probably, the H5,7DHF anion links to Cu(II) in a similar manner, while the 7,8DHF e" anion must interact with Cu(II) through the contiguous deprotonated OH.
The electronic spectra of all the copper(II) complexes exhibit in the solid state one major absorption band in the 12500-20000 cm 1 region. This band is attributed to d-d transitions in an octahedral geometry or a square pyramidal distorted structure. One band at 22500-24500 cm " is attributed to a charge-transfer transition from the flavonolato ligand to the metal atom. Furthermore, the complexes show an intraligand transition around 42000 cm.
The values of the magnetic moments at room temperature of the copper complexes of the H3HF and H25,7DHF are in the normal range for Cu(II) complexes without metal-metal interaction (1.74-1.93 MB). The magnetic moment values at room temperature of Cu(7,8DHF)(H20): and Cu(7,8DHF)(MeOH) are 1.43 and 1.40 MB respectively, suggesting metal-metal antiferromagnetic interaction. The complex Cu (  The progression of the cleavage reaction with MPA (mercaptopropionic acid) was also examined. Figure 3 shows that the complex is a chemical nuclease at 50 and 75 ktM, but as we compare with that of H202 we can deduce a lower nuclease activity of the Cu(7,8DHF)(NHa)2(H20)3 complex in the presence of We tried to study the cleavage reaction of pBR322 in the presence of the other complex, Cu(7,SDHF)(H.,O)2 in the same conditions as reported above. Figure 4 confirms the chemical nuclease activity of this complex in the presence of H202 or MPA or ascorbic acid. From this figure we can observe that the complex only shows nicked and linear forms in the complex concentration range from 20 to 40 tM.
If we compare its nuclease activity in presence of H202 or MPA or ascorbic acid, we can deduce that the increasing activity order is ascorbic acid > H202 > MPA. 1   In order to assess some specificity, supercoiled pUCI plasmid DNAwas cleaved with the Cu(7,SDHF)MeOH, followed by probing with EcoRI. (Figure 6). When we compare lanes and 2 that correspond to a result of the cleavage in the presence of the complex (lane 1) and afterwards digested with EcoRI (lane 2), the existence of three bands in the latter can suggest a preferential cleavage of the Cu(7,SDHF)(MeOH) complex. From these experiences we can conclude that the Cu(7,SDHF)(NH3)z(HzO)3, Cu(7,8DHF)(HzOh and Cu(7,8DHF)(MeOH) complexes are chemical nucleases. We also plat, to continue to study the possible specificity of the complex and the other related ones in the hope of discovering other potentially practical inorganic nucleases. Involvement of the Cu(1) in the reaction.-We employed bathocuproine as an agent that sequesters Cu(1) selectively. The Cu(l)-bathocuproine chelate has an absorption maximum at 480 nm. Under the conditions of our reaction, neither Cu(ll) nor Hz7,8DHF interferes with the absorption maximum, whereas Hz7,SDHF and Cu(II) react to generate Cu(1) that in the presence of bathocuproine, gives rise to the maximum at 480 nm (Figure 7). The implication of these findings is that Cu(II) is reduced by H27,8DHF, which does not establish whether Cu(1) is an end-product or an intermediate in the reaction that occurs in the absence of sequestering compound. As we obtain the solid complexes, we think that the Cu(I) is an intermediate that it is oxidised to Cu(II) and reacts to H27,8DHF in order to form the complexes. Our data establish that the copper complexes of H27,8DHF generate single-strand and double strand breaks in the presence of reducing agents such as ascorbic acid, H2Oz or MPA. These complexes have more nuclease activity than the mixtures of Cu(II) and Hz7,8DHF. If we observe the chemical formula of H7,8DHF, we can observe the presence of two OH contiguously. Hadi 'z reported the nuclease activity of the quercetin and other flavonoids in the presence of Cu(II) and oxygen and concluded that the presence of two OH groups in positions 3' and 4' of the B ring is required for this activity. Our results indicate that the two contiguous OH are necessary but they can occupy positions in the A ring also.
According the mechanisms proposed previously by Sigman3 and Hadi '" for copper nucleases, we suggest a similar one, where the flavone reduces the Cu(lI) to Cu(I) after that the complex is intercalated to DNA and then reacts to HOz to give rise oxygen radical species and an oxidised form of Hz7,8DHF, that may be a group of compounds. The radicals are the responsible for the DNA cleavage.