Velocidad en relación con los puntos ajustados

UV-Vis studies have been carried out to investigate the ability of the terpy ligand to bind copper (Figure 8.2). The spectra obtained are comparable to previously published spectra collected for both copper and platinum terpy complexes.[33] _{The initial spectrum of terpy changes} drastically upon addition of CuCl2. The differences in the spectra of 0.5 equivalents of copper and 1 equivalents of copper are distinct. For example, a strong increase in absorption is observed at 220 nm with increasing copper concentration. Moreover, the sharp peak at 280 nm is observed only for 1 or more equivalents of copper, and not for 0.5 equivalents of copper per terpy ligand. The spectrum does not change so drastically upon addition of more than one equivalent of CuCl2 to the terpy ligand, although marked differences are observed at 260 and 220 nm. Nevertheless, the complexes Cu(terpy), [Ru(dtdeg)Cu], [Cu2(dtdeg)2Ru], [Cu2(dtdeg)3Ru2],

[Cu(dtdeg)Ru(bipy)Cl] and [Cu(dtdeg)RuCl3] have been prepared in situ with one equivalent

of copper per terpy ligand throughout the whole study presented in this chapter, since no differences in cleaving activity have been observed with one, or one and a half equivalents of copper per terpy unit.

0 200 0.5 1 1.5 2 250 300 350 400 Wavelength (nm) Ab so rp tion no copper 0.5 equiv copper 1 equiv copper 1.5 equiv copper 2 equiv copper 150

DNA cleavage by heteronuclear Ru/Cu complexes Figure 8.2 Titration of CuCl2 to

nt quantities of copper have been usedthe ligand terpy (50

µM) followed by UV-Vis spectroscopy. : 0.5, 1, 1.5 and 2 equivalents per 1 equivalent f terpy ligand.

Frozen EPR spectra have been recorded of the complexes Cu(terpy), [Ru(dtdeg)Cu], u₂(d

Four differe o

[C tdeg)₂Ru], [Cu₂(dtdeg)₃Ru₂], [Cu(dtdeg)Ru(bipy)Cl] and [Cu(dtdeg)RuCl₃] in H₂O. It is shown that a part of the Cu(II) of the Cu(terpy) and [Cu(dtdeg)RuCl₃] has dissociated from the ligand to the form the Cu(H₂O)₆2+ _{complex. Nevertheless, unique peaks are observed} that correspond with Cu(II) binding to nitrogen ligands. The recorded EPR signals of the other complexes are too broad, because the freezing of H₂O solution of the complexes did not lead to a good glass. It is assumed that the other complex solutions have similar behavior, since the binding unit for the copper is the same.

Chapter 8 Figure

u(dtdeg)Ru(bipy)Cl] and [Cu(dtdeg)RuCl₃] has been investigated and compared. The concentrations shown in this chapter are of the complexes, therefore [Cu₂(dtdeg)₂Ru] and [Cu₂(dtdeg)₃Ru₂] have a two times higher copper concentration compared to the other complexes. The ruthenium compounds in combination with other metal ions, like Fe, Ni, Co and Mn do not show any nuclease activity. DNA is reacted for 1 hour with the complexes and mercaptopropionic acid (MPA) prior to loading the samples on agarose gel. The optimal cleavage conditions are obtained with phosphate buffer (pH 7.2) and MPA, because significantly lower cleavage activities are observed in HEPES buffer (pH 7.2) and with ascorbate acid as reductant. All the experiments have been performed reproducibly with phosphate buffer and MPA as added reductant.

The activity of each complex has been tested at four different concentrations (1, 2, 5 and 10 µM; Figure 8.3). The complexes do not show any nuclease activity in the absence of copper or MPA. With copper and MPA activity is observed, and the following cleavage activity sequence has been found: [Cu₂(dtdeg)₃Ru₂] > [Cu₂(dtdeg)₂Ru] ≈ [Cu(dtdeg)Ru(bipy)Cl] > [Ru(dtdeg)Cu] >> Cu(terpy) ≥ [Cu(dtdeg)RuCl₃]. It should be noted that [Cu₂(dtdeg)₃Ru₂] and [Cu₂(dtdeg)₂Ru] have two active copper units compared to the other complexes. The nuclease activity of Cu(terpy) is low at 2 µM, because only a small fraction of supercoiled DNA is converted to circular DNA. At higher complex concentrations, linear DNA is also formed and a smear appears at a concentration of 10 µM. Compared to Cu(terpy), the complex [Ru(dtdeg)Cu], including one ruthenium moiety and one copper unit, is substantially more active per copper ion. At a complex concentration of 2 µM, the entire supercoiled DNA is transformed to circular and linear DNA. At 10 µM, all DNA is cut into relatively small fragments that cannot be visualized by their treatment with ethidium bromide. Logically, the complex that contains two copper units and one ruthenium moiety shows an increased nuclease activity compared to [Ru(dtdeg)Cu] and Cu(terpy). A smear (multi-fragmented DNA) already appears at a [Cu₂(dtdeg)₂Ru] concentration of 5 µM. At a concentration of 1 µM, the supercoiled DNA is totally converted to mainly circul d, to a lesser extent, to linear DNA. The most

ot detectable at a 8.3 DNA cleavage activity of Cu(terpy), [Ru(dtdeg)Cu], [Cu2(dtdeg)2Ru],

[Cu2(dtdeg)3Ru2], [Cu(dtdeg)Ru(bipy)Cl] and [Cu(dtdeg)RuCl3] (see scheme 1 for complex

details). Lane 1 : complex concentration of 1 µM. Lane 2 : complex concentration of 2 µM. Lane 3 : complex concentration of 5 µM. Lane 4: complex concentration of 10 µM.

8.2.2 Cleavage of supercoiled DNA

The relaxation of supercoiled circular ΦX174 DNA (form I) into the relaxed (form II) and the linear (form III) conformations mediated by the complexes Cu(terpy), [Ru(dtdeg)Cu], [Cu₂(dtdeg)₂Ru], [Cu₂(dtdeg)₃Ru₂], [C

ar DNA an

active cleaving agent [Cu₂(dtdeg)₃Ru₂] has two ruthenium units and two copper centers. A large fraction of linear DNA (Form III) is observed at a complex concentration of 1 µM. A smear starts to appear at a concentration of 2 µM and the three forms of DNA are n

DNA cleavage by heteronuclear Ru/Cu complexes complex concentration of 5 µM. The complex [Cu(dtdeg)Ru(bipy)Cl] exhibits a different

thenium unit, consisting of a ruthenium(ΙΙΙ) ion coordinated by one terpy and one bipy ligands and a c

2 2 3 2

3 er investigate their cleaving ability (Figure 8.4).

The ma

o ru

hloride anion. Interestingly, this complex shows a nuclease activity (at similar complex concentrations) comparable to the one of [Cu2(dtdeg)2Ru] at similar complex concentrations

even though it holds only one copper unit. Probably, the interaction of the ruthenium unit of [Cu(dtdeg)Ru(bipy)Cl] with DNA is stronger than the one of the Ru moiety of [Cu2(dtdeg)2Ru]. The neutral complex [Cu(dtdeg)RuCl3]is less active than Cu(terpy). This

difference in reactivity is clearly observed at 10 µM, where the action of [Cu(dtdeg)RuCl3]

produces a smear, while Cu(terpy) does not. The ruthenium unit of [Cu(dtdeg)RuCl3]is not

charged and therefore its interaction with DNA is less favored compared to the charged complexes [Ru(dtdeg)Cu], [Cu2(dtdeg)2Ru] and [Cu2(dtdeg)3Ru2], [Cu(dtdeg)Ru(bipy)Cl].

Time-course studies of DNA cleavage mediated by the complexes Cu(terpy), [Ru(dtdeg)Cu], [Cu2(dtdeg) Ru], [Cu (dtdeg) Ru ], [Cu(dtdeg)Ru(bipy)Cl] and

[Cu(dtdeg)RuCl ] have been carried out to furth

jor difference is the use of larger volumes of reactant solutions (see experimental section) for the kinetic studies (compared to the typical experiments), which may result in small discrepancies regarding the cleavage activities. These experiments confirm the sequence of cleavage ability determined earlier. It has to be noticed that [Cu2(dtdeg)2Ru] and

[Cu(dtdeg)Ru(bipy)Cl] show lower cleaving activities at the same complex concentrations, using these experimental conditions compared to the results shown in Figure 8.3. All plots illustrate a decrease of Form I with concomitant increase of Form II. Form III is not formed, or only in a slight amount at a later stage of the cleavage reaction, and only after all Form I is converted. In all cases, less than 10 % of Form III is formed, at the end of the cleavage reactions. All the complexes display the typical behavior of single-strand cleaving agents. Indeed, the action of double strand cleaving agents is characterized by the production of Form III directly from the beginning of the cleavage reaction (see for example the mechanism of action of the complexes reported in chapters 3 and 4).[34]

The nature of the active species responsible for the cleavage of DNA can be investigated by the addition f inhibitors. So, NaN3, superoxide dismutase, DMSO or ethanol have been added to the reaction mixture containing the complexes Cu(terpy), [Ru(dtdeg)Cu], [Cu2(dtdeg)2Ru], [Cu2(dtdeg)3Ru2], [Cu(dtdeg)Ru(bipy)Cl], or [Cu(dtdeg)RuCl3] to

uncover the active species of the cleavage reaction. Cleavage reactions have also been performed under argon, pure dioxygen and in the dark. NaN3 is able to quench singlet oxygen, superoxide dismutase is able to scavenge superoxide radicals, and DMSO and ethanol are known to neutralize hydroxyl radicals. The reaction under argon reveals an active role of dioxygen in the reaction medium. The reactions carried out in the dark reveal the potential of the complexes to perform photocleavage of DNA. If dioxygen is involved in the cleavage process and its binding

Chapter 8

to the complex is the rate-determining step, then the amount of DNA cuts would be expected to increase under an atmosphere of dioxygen.

Figure 8.4 Time-course experiments of DNA cleavage (20 µM base pairs) over a period of 70 minutes in the presence of 5 mM MPA and air. The different plots have been obtained using, respectively, 5 µM Cu(terpy), 2 µM [Ru(dtdeg)Cu], 2 µM [Cu2(dtdeg)2Ru], 1 µM

[Cu2(dtdeg)3Ru2], 3 µM [Cu(dtdeg)Ru(bipy)Cl] and 8 µM [Cu(dtdeg)RuCl3].

Cleavage reactions in the presence of scavengers have been investigated for all complexes. However, only the results obtained with [Ru(dtdeg)Cu] are depicted in Figure 8.5, because all the compounds give comparable results suggesting very similar mechanisms. In Figure 8.5, lane 1 illustrates the aerobic cleavage reaction mediated by [Ru(dtdeg)Cu] in the presence of MPA (normal cleavage conditions). The reactions of [Ru(dtdeg)Cu] with DNA in the presence of NaN3, superoxide dismutase, DMSO, ethanol, under dioxygen, or in the dark are similar to the one performed under normal cleavage conditions. In contrast, the reaction under argon is almost completely inhibited, which indicates that dioxygen is involved in the cleavage reaction. The

DNA cleavage by heteronuclear Ru/Cu complexes active species generated from Cu(terpy), [Ru(dtdeg)Cu], [Cu2(dtdeg)2Ru], [Cu2(dtdeg)3Ru2],

[Cu(dtdeg)Ru(bipy)Cl] and [Cu(dtdeg)RuCl3] is therefore most likely a copper bound

oxidant, which is not affected by the presence of any of the used scavengers.

Figure 8.5 Cleavage reactions mediated by [Ru(dtdeg)Cu] in the presence of various scavengers performed in air (except lane 4). All experiments were performed with a complex concentration of 5 µM. Lane 1: no extra additives. Lane 2: 100 µM NaN3. Lane 3: 0.5 units superoxide dismutase. Lane 4: under argon. Lane 5: in the dark. Lane 6: under dioxygen. Lane 7: 20 µM DMSO. Lane 8: 20 µM ethanol.

The ruthenium units are probably able to interact with DNA by electrostatic interaction and partial intercalation. An increase of the ionic strength of the reaction medium is expected to reduce the affinity of such compounds for DNA. Therefore, experiments have been performed with increasing concentrations of NaCl. Only the cleavage products obtained with Cu(terpy) and [Ru(dtdeg)Cu] are shown in Figure 8.6, because the use of complexes [Cu2(dtdeg)2Ru],

[Cu2(dtdeg)3Ru2], [Cu(dtdeg)Ru(bipy)Cl] and [Cu(dtdeg)RuCl3] gives analogous results

compared to [Ru(dtdeg)Cu]. The cleaving activities of all complexes start to decrease from a

is diminished at a NaCl concentration of 300 mM, but the nuclease activity is not quenched. This NaCl concentration of 50 mM and above (Lanes 4 and 12, Figure 8.6). At 300 mM NaCl, no cleaving activity is observed for [Ru(dtdeg)Cu], [Cu2(dtdeg)2Ru], [Cu2(dtdeg)3Ru2],

[Cu(dtdeg)Ru(bipy)Cl] and [Cu(dtdeg)RuCl3]. The cleaving activity of Cu(terpy)

result indicates that Cu(terpy) interacts in a different manner with DNA, compared with the complexes that include a ruthenium moiety. Its interaction with DNA is apparently dominated by partial intercalation of the terpy ligand, which is not perturbed by an enhancement of the ionic strength of the solution.

Figure 8.6 Influence of the ionic strength on the cleavage abiliti

µ es of Cu(terpy)

and[Ru(dtdeg)Cu]. Lanes 1-8: Cu(terpy) (5 M). Lanes 9-16: [Ru(dtdeg)Cu] (2 µM). Lanes 1

Chapter 8

and 9: no NaCl. Lanes 2 and 10: 10 mM NaCl. Lanes 3 and 11: 20 mM NaCl. Lanes 4 and 12: 50 mM NaCl. Lanes 5 and 13: 100 mM NaCl. Lanes 6 and 14: 300 mM NaCl. Lanes 7 and 15: 500 mM NaCl. Lanes 8 and 16: 625 mM NaCl.

The ruthenium unit in [Cu(dtdeg)Ru(bipy)Cl] and [Cu(dtdeg)RuCl3] has the

possibility to coordinate to DNA, because the complexes have respectively one and three chloride ligands. As mentioned in previous chapters, the complexes have been pre-incubated with DNA for 24 hours before the initiation of the cleavage reaction. However, no difference in

u(dtdeg)RuCl3] have been treated with, respectively,

nuclease activity between the experiments with and without pre-incubation time is observed. In the following experiment, the DNA was precipitated after the pre-incubation time, prior to the cleavage reaction, to remove any unreacted complex. Interestingly, the complexes do not show any DNA cleavage under these experimental conditions. To increase their reactivity towards DNA, [Cu(dtdeg)Ru(bipy)Cl] and [C

one and three equivalents of AgNO3 to remove the chloride ligands. This method is commonly used for replacing a chloride by a water ligand in order to enhance the reactivity of a platinum center towards nucleophiles. After a pre-incubation time of 24 hours and precipitation of DNA or not, the DNA cleavage reaction is initiated (Figure 8.7). Both complexes are less efficient cleaving agents after treatment with AgNO3 and pre-incubation time of 24 hours. It is expected that the complexes would have a stronger DNA interaction after removal of the chlorides and thus higher nuclease activity. However, the nuclease activity is lower after AgNO3 treatment. The explanation for these surprising results is not yet available. Without precipitation step, [Cu(dtdeg)Ru(bipy)Cl] (lanes 1 and 2, Figure 8.7) and [Cu(dtdeg)RuCl3] (lanes 5 and 6,

Figure 8.7) display cleavage activities. With a precipitation step no cleavage is observed for both complexes (Lanes 3, 4, 7, and 8, Figure 8.7). These results indicate that the two complexes, despite preactivation, do not coordinate to supercoiled DNA under these experimental conditions.

Figure 8.7 DNA Cleavage experiments of [Cu(dtdeg)Ru(bipy)Cl] and [Cu(dtdeg)RuCl3]

after reaction with AgNO₃ and a pre-incubation time of 24 hours. Lane 1: 5 µM

step. Lane 8: 10 µM [Cu(dtdeg)RuCl3], with

precipitation step.

[Cu(dtdeg)Ru(bipy)Cl], no precipitation step. Lane 2: 10 µM [Cu(dtdeg)Ru(bipy)Cl], no precipitation step. Lane 3: 5 µM [Cu(dtdeg)Ru(bipy)Cl], with precipitation step. Lane 4: 10

µM [Cu(dtdeg)Ru(bipy)Cl], with precipitation step. Lane 5: 5 µM [Cu(dtdeg)RuCl3], no

precipitation step. Lane 6: 10 µM [Cu(dtdeg)RuCl3], no precipitation step. Lane 7: 5 µM

[Cu(dtdeg)RuCl3], with precipitation

DNA cleavage by heteronuclear Ru/Cu complexes