CHAPTER IV

CHAPTER IV

Acridines: D and ZnD interaction with DNA

Acridines (among them proflavine) are a special class of compounds not only because of their wide use in the pharmaceutical and dye industries but also due to their interesting chemical and physical properties (Albert, 1966; Zittoun, 1985). They were the first chromophores whose non covalent interactions with DNA were extensively studied exploiting their intense fluorescence and now their binding to double strands is almost universally interpreted according to intercalation (Lerman, 1961; Aggarwal et al., 1984; Adams, 2002; Malinina et al., 2002). Numerous studies have shown that the intercalative process is much more complex than it was supposed to be and that its features heavily depend on the structure of the intercalating molecule (Wakelin and Waring, 1980). In this context, bifunctional molecules bearing both an aromatic moiety and a separate metal complexing centre appear to be of particular interest (Lippard, 1978; Hiort et al., 1993; Arounaguiri et al., 2000). The aromatic residue provides an anchorage for the molecule on the polymer chain by intercalation, whereas the metal centre may intercalate as well or interact with the polymeric backbone, there exerting a given function (Krotz et al., 1993; Terbrueggen and Barton, 1995). Barton and co-workers (Erkkila et al., 1999) have synthesized special metallointercalators with a diversity of functions, from luminescent probes for DNA (Olson et al., 1997) to structural probes for RNA (Lim and Barton, 1997). Other complexes have been designed, able to recognize specific sites on DNA as base pair mismatches (Jackson and Barton, 1997; Jackson et al., 1999). Moreover, metallointercalators able to promote the cleavage of the phosphodiester bond at a selected point of the chain length have been prepared. Platinum(II) complexes appended to an aromatic residue are other special metal intercalators. They are synthesised focussing on their possible anticancer activity (Wakelin and Waring, 1980; Marzilli et al., 1999; Boudreaux, 2001). Intercalative binding of these complexes to nucleic acids constitute a prerequisite for the subsequent slow attack of Pt(II) to the base nitrogen (Bowler et al., 1989; Ciatto et al., 1999).

On the basis of the above mentioned arguments, we found it interesting, in the framework of this thesis, to investigate the interaction of a fluorescent metallointercalator with double stranded calf thymus DNA. This molecule, shown in Fig. 4.1 and from now on denoted as D (dye), has been synthesised in the

University of Florence by the group of Prof. Antonio Bianchi with whom we collaborate. The bifunctional molecule is characterized by a proflavine unit linked to a diethylenetriamine moiety, able to chelate metal ions. The novel structural aspect of D consists in the fact that the metal binding site is linked to the C9 position of the proflavine instead of N10 as in similar metallointercalators (Bowler et al., 1989). At physiological pH the N10 atom of D is expected to be protonated in both the metal-free ligand and its metal complexes; this study can therefore enlighten the effect of a positively charged N10 ammonium group on the DNA binding process. Accordingly, the metal complex forming ability of D and the thermodynamic and kinetic features of the interaction of D and its Zn(II) complex with DNA have been investigated. At pH = 7.0 the dye, in its three-protonated

form [H3D]3+, is almost the unique species in solution. Measurements on the

interaction of DNA with the Zn(II) complex of D, in 1:1 molar ratio, were

performed at pH = 8.0 where the complex [ZnD]2+ _{is majority. The high stability}

constant of this specie ensures that [ZnD]2+ _{is completely formed when reacting}

with DNA (Bazzicalupi et al., 2007). From now on D will denote the species [H3D]3+

and ZnD the specie [ZnD]2+_.

N NH2 NH2 HN N NH2 NH2 A _{N NH} 2 NH2 HN N NH2 NH2 Zn2+ B

Fig. 4.1. (A) Structure of the bifunctional dye 3,6-diamine-9-[6,6-bis(2-aminoethyl)-1,6-diaminohexyl]acridine, denoted as D in the text. The aromatic residue is a proflavine dye, whereas the metal coordinating moiety is a polyamine. (B) ZnD complex.

4.1

Spectral analysis of aggregation

It is known that acridine derivatives give rise to self-aggregation in aqueous solution with the formation of dimers, or larger oligomeric species at higher concentrations (Glazer, 1965; Lamm and Neville, 1965; Blears and Danyluk, 1967; Chambers et al., 1974; Porumb, 1978; Bowler et al., 1989; Luchowski and Krawczyk, 2003). Since the aggregation process can interfere with all other equilibria, the ability of D and its Zn(II) complex to form aggregates was first investigated.

The analysis of D and ZnD aggregation was performed by recording several absorbance spectra for different dye concentrations. The shape of the absorbance spectra does not exhibit any noticeable change on increasing dye concentrations and no additional bands appear at high dye levels (Figure 4.2A). This behaviour seems to indicate that no dye self-aggregation occurs in the investigated

concentration range (0 ÷ 8.9×10-4_{M). This finding was confirmed by a check of the}

linearity range of an absorbance vs. dye concentration plot. For comparison the analysis was made also for the proflavine dye alone, which is known to undergo self-aggregation. Deviations from the linearity, indicating aggregate formation, occur only for proflavine (Fig 4.2B). These features indicate that both proflavine derivates ZnD and D do not self-aggregate in the investigated range of (relatively low) concentrations. 200 300 400 500 0 1 2 A λ(nm)

A

B

Fig. 4.2. (A) Absorbance spectra of D from CD = 0 to CD = 8.9×10-4M (top). I = 0.10 M (NaCl),

pH = 7.0, T = 25°C (optical path = 0.1 cm). The spectral behaviour seems to indicate absence of self-aggregation processes. (B) Absorbance as a function of dye concentration, CD. (•)

proflavine λ = 444 nm, () ZnD λ = 390 nm and (■) D λ = 390 nm, I = 0.10 M (optical

path = 0.1 cm).

4.2

NMR measurements

If spectrophotometric results indicate that no aggregation occurs in the diluted concentration range, NMR experiments performed in Florence, however, suggest

dimer formation for concentrations of D and ZnD2+ _{higher than 1×10}-3_M

(Bazzicalupi et al., 2007).

1_{H NMR spectra in D2O recorded at different pH values and with D concentration}

greater than 1×10-3 _{M showed that in alkaline solutions (pH > 8.5) all resonances}

are split (Fig. 4.3). This second set of signals reflects the formation of a second equilibrium species, having the same symmetry of the parent one and whose

interconversion is slow on the NMR time scale. 1_{H NMR measurements performed}

with different ligand concentrations (0.01-0.001 M) and at different temperatures (25 - 55°C) evidenced that the abundance of the new species increases with increasing ligand concentration and decreases with increasing temperature, while an opposite trend is found for the other species. These data are in agreement with a dimeric nature of the new species in equilibrium with monomeric D. Dye molecules associate forming π-stacking interactions between the heteroaromatic units, as denoted by the significant upfield shift experienced by the acridine

protons (δH1-δH1’ = 0.096 ppm, δH2-δH2’ = 0.046 ppm, δH3-δH3’ = 0.042 ppm at pH 10,

Fig. 4.3). Furthermore, the interconversion between the two species is reversible with respect to changes of pH, concentration, and temperature.

7.80 7.40 7.00 6.60 6.20 5.80 ppm 1 ₂ 3 3.80 3.40 3.00 2.60 2.20 1.80 ppm 3.60 3.20 2.80 2.40 2.00 4 7 6 5 7.80 7.40 7.00 6.60 6.20 5.80 ppm 1 ₂ 3 3.80 3.40 3.00 2.60 2.20 1.80 ppm 3.60 3.20 2.80 2.40 2.00 4 7 6 5 7.80 7.40 7.00 6.60 6.20 5.80 ppm 1 2 3 1 ₂ 3 4 4 7 7 6 6 5 5 3.80 3.60 3.40 3.20 3.00 2.80 2.60 2.40 2.20 2.10 1.80 ppm 7.80 7.40 7.00 6.60 6.20 5.80 ppm 1 2 3 1 ₂ 3 4 4 7 7 6 6 5 5 3.80 3.60 3.40 3.20 3.00 2.80 2.60 2.40 2.20 2.10 1.80 ppm 1 2 3 4 5 6 7 N NH2 H2N HN N NH2 NH2 pH= 4 pH=1 0 1_{H NMR} (D 2O, 298.1K) CL=1.0 6 ·10- 2_M 2L L2 7.80 7.40 7.00 6.60 6.20 5.80 ppm 1 ₂ 3 3.80 3.40 3.00 2.60 2.20 1.80 ppm 3.60 3.20 2.80 2.40 2.00 4 7 6 5 7.80 7.40 7.00 6.60 6.20 5.80 ppm 1 ₂ 3 3.80 3.40 3.00 2.60 2.20 1.80 ppm 3.60 3.20 2.80 2.40 2.00 4 7 6 5 7.80 7.40 7.00 6.60 6.20 5.80 ppm 1 2 3 1 ₂ 3 4 4 7 7 6 6 5 5 3.80 3.60 3.40 3.20 3.00 2.80 2.60 2.40 2.20 2.10 1.80 ppm 7.80 7.40 7.00 6.60 6.20 5.80 ppm 1 2 3 1’ _2’ 3’ 4 4’ 7 7’ 6 6’ 5 5’ 3.80 3.60 3.40 3.20 3.00 2.80 2.60 2.40 2.20 2.10 1.80 1 2 3 4 5 6 7 N NH2 H2N HN N NH2 NH2 pH 4 pH 10 7.80 7.40 7.00 6.60 6.20 5.80 ppm 1 ₂ 3 3.80 3.40 3.00 2.60 2.20 1.80 ppm 3.60 3.20 2.80 2.40 2.00 4 3.80 3.40 3.00 2.60 2.20 1.80 ppm 3.60 3.20 2.80 2.40 2.00 4 7 6 5 7.80 7.40 7.00 6.60 6.20 5.80 ppm 1 ₂ 3 3.80 3.40 3.00 2.60 2.20 1.80 ppm 3.60 3.20 2.80 2.40 2.00 4 7 3.80 3.40 3.00 2.60 2.20 1.80 ppm 3.60 3.20 2.80 2.40 2.00 4 7 6 5 7.80 7.40 7.00 6.60 6.20 5.80 ppm 1 2 7.80 7.40 7.00 6.60 6.20 5.80 ppm 1 2 3 1 ₂ 3 4 4 7 7 6 6 5 5 3.80 3.60 3.40 3.20 3.00 2.80 4 4 7 7 6 6 5 5 3.80 3.60 3.40 3.20 3.00 2.80 2.60 2.40 2.20 2.10 1.80 ppm 7.80 7.40 7.00 6.60 6.20 5.80 ppm 1 2 7.80 7.40 7.00 6.60 6.20 5.80 ppm 1 2 3 1 ₂ 3 4 4 7 7 6 6 5 5 3.80 3.60 3.40 3.20 3.00 2.80 4 4 7 7 6 6 5 5 3.80 3.60 3.40 3.20 3.00 2.80 2.60 2.40 2.20 2.10 1.80 ppm 1 2 3 4 5 6 7 N NH2 H2N HN N NH2 NH2 pH= 4 pH=1 0 1_{H NMR} (D 2O, 298.1K) CL=1.0 6 ·10- 2_M 2L L2 7.80 7.40 7.00 6.60 6.20 5.80 ppm 1 ₂ 3 3.80 3.40 3.00 2.60 2.20 1.80 ppm 3.60 3.20 2.80 2.40 2.00 4 7 3.80 3.40 3.00 2.60 2.20 1.80 ppm 3.60 3.20 2.80 2.40 2.00 4 7 6 5 7.80 7.40 7.00 6.60 6.20 5.80 ppm 1 ₂ 3 3.80 3.40 3.00 2.60 2.20 1.80 ppm 3.60 3.20 2.80 2.40 2.00 4 7 6 5 7.80 7.40 7.00 6.60 6.20 5.80 ppm 1 2 7.80 7.40 7.00 6.60 6.20 5.80 ppm 1 2 3 1 ₂ 3 4 4 7 7 6 6 5 5 3.80 3.60 3.40 3.20 3.00 2.80 4 4 7 7 6 6 5 5 3.80 3.60 3.40 3.20 3.00 2.80 2.60 2.40 2.20 2.10 1.80 ppm 7.80 7.40 7.00 6.60 6.20 5.80 ppm 1 2 7.80 7.40 7.00 6.60 6.20 5.80 ppm 1 2 3 1’ _2’ 3’ 4 4’ 7 7’ 6 6’ 5 5’ 3.80 3.60 3.40 3.20 3.00 2.80 2.60 2.40 2.20 2.10 1.80 1 2 3 4 5 6 7 N NH2 H2N HN N NH2 NH2 pH 4 pH 10

Fig. 4.3. 1_{H NMR spectra of D (1×10}-2 _{M) at pH 4.0 and pH 10.0. Primed labels refer to}

dimeric species.

4.3

D and ZnD interaction with DNA

4.3.1 Equilibria

On the basis of the previous results, appropriate concentrations of dyes (1×10-5 _M

for absorbance titrations and 1×10-6 _{M for fluorescence titrations) were selected to}

ensure the absence of dye aggregates in the solutions used to study the interaction of D and its Zn(II) complex with DNA. The interaction is revealed by changes in the fluorescence and absorbance spectra occurring when increasing amounts of DNA are added to a dye solution.

A fluorescence emission study performed with the metal-free ligand at pH 7.0,

where [H3D]3+ is almost the unique specie in solution, showed a sharp decrease of

the emission intensity upon the early additions of DNA to D (phase (1)) followed by an intensity increase (phase (2)) and a shift of the emission maximum towards smaller wavelengths (Figure 4.4A). Figure 4.4B shows a fluorescence titration

performed at I = 0.10 M, pH = 7.0, λexc = 385 nm, λem = 500 nm and T = 25 °C.

The binding isotherm points out the biphasic behaviour of the system, which reveals the occurrence of two different modes of interaction.

400 450 500 550 600 0 100 200 300 A (2) F λ(nm) (1) 0.0 0.5 1.0 1.5 -2.0 -1.5 -1.0 -0.5 0.0 (2) (1)

B

Fig. 4.4. Spectrofluorometric analysis of the binding of D to DNA; CD = 1×10-6 M, I = 0.10 M

(NaCl), pH = 7.0, T = 25°C, λexc = 385 nm. (A) Fluorescence spectra collected during titration;

the spectral changes reveal the biphasic behaviour of the binding process. (B) Binding isotherm at λem = 500 nm; the two phases, (1) and (2), are associated to two different

binding modes.

The interaction of DNA with the Zn(II) complex of D has been investigated as well.

The measurements were performed at pH 8.0 where the complex [ZnD]2+ _{is the}

unique species in solution. The high stability constant of this species, determined by means of potentiometric titrations (Bazzicalupi et al., 2007), ensures that the complex is completely formed prior to reaction with DNA and the metal ion is retained by D; thus, the spectral changes observed after mixing the preformed complex with a solution of DNA only reflect the occurrence of a polymer-dye binding reaction.

The spectral behaviour of the DNA/ZnD system is similar to that of the DNA/D system and the features of the binding isotherms provide evidence for biphasic behaviour of this system as well (Fig. 4.5A and B).

420 460 500 540 580 0 90 180 270 360 (2) F λ (nm) A (1) 0.0 0.5 1.0 1.5 2.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 10 -8 ∆ F/ C Zn D (M -1 ) 104_C P (M)

B

Fig. 4.5. (A) Fluorescence spectra for the DNA/ZnD system. CD = 1×10-6 M, I = 0.10 M

(NaCl), pH = 8.0, T = 25°C. The fluorescence emission decreases in the binding phase (1) and increases in the binding phase (2). (B) The biphasic binding isotherm recorded at

λ = 500 nm.

Absorbance titrations confirm the findings described above. Absorbance spectra of the DNA/D system, recorded during titrations, show that absorbance first decreases (phase (1)) and then increases (phase (2)) displaying a bathochromic shift (Fig. 4.6A). A similar feature is exhibited by DNA/ZnD system (Fig. 4.6B).

300 400 500 0.0 0.1 0.2

A

_B

Fig. 4.6. Absorbance spectra collected during titration for the DNA/D (A) and DNA/ZnD (B) systems; I = 0.10 M (NaCl), T = 25°C. (A) CD = 1×10-5 M and CP = 0 ÷ 2.5×10-4 M, pH = 7.0;

(B) CZnD = 1×10-5 M and CP = 0 ÷ 2.0×10-4 M, pH = 8.0. The spectral changes reveal the

biphasic behaviour of the binding process. The two phases, (1) and (2), are associated to two different binding modes.

From now on we shall indicate the first branch of titration curves (decrease of fluorescence and absorbance) as binding mode 1 and the second branch of the curve (enhancement of fluorescence and absorbance) as binding mode 2.

The binding process can be again represented by the apparent reaction (4.1) (4.1) where the dye (D) interacts with the DNA sites (S) to give the bound species (DS). The equilibrium constant is defined as K = [DS]/([D]×[S]).

In order to evaluate the binding parameters of the two binding modes, the two branches of titration curves were analysed according to eq. (4.2)

) KC (1 C K∆ C ∆F P P D + × φ = (4.2)

where CD and CP are the total dye and polymer concentrations respectively;

∆F = F - φDCD is the change of fluorescence (F) during titration, and φD = F°/CD,

where F° denotes the initial fluorescence of the dye solution; ∆φ = φPD - φD is the

amplitude of the binding isotherm. The analysis of the titration curves allows

evaluating K and ∆φ for the two binding modes (K1, ∆φ1 for biding mode 1 and K2,

∆φ2 for binding mode 2). The values of K1 and K2 are collected in Table 4.1. The

quality of fit is shown in Figure 4.4B, where the continuous line is calculated using eq. (4.2). Note that in eq. (4.2) the total polymer concentration appears in place of the more correct free site concentrations [S] (see Appendix II). This simplification is justified since in fluorescence measurements the conditions

CP>>100CD holds and under these circumstances the variable f(r) which links [S]

to CP ([S] = CPf(r)) is almost unity.

4.3.2 Salt concentration dependence of equilibria

Fluorescence titrations have been carried out at different added salt

concentrations. Since D ([H3L]3+) and ZnD ([ZnD]2+) are positively charged, a salt

dependence of the equilibria is likely to occur. In effect, the binding isotherms of the DNA/D system show a dependence on ionic strength. At I = 0.01 M and I = 0.10 M the biphasic nature of the binding process is well evident, whereas at I = 1.0 M a monophasic behaviour is substantially observed (Fig. 4.7A).

Concerning the DNA/ZnD system the two modes of binding are present also at I = 1.0 M (Fig. 4.7B), revealing that the metal ion plays a role in the interaction of the zinc-complex with DNA. Table 4.1 summarizes the results.

Table 4.1. Reaction parameters for the interaction of CT-DNA with D and ZnD, T = 25°C.

(a) (Biver et al., 2003), (b) (Li and Crothers, 1969), (c) (Dourlent and Hogrel, 1976)

0.0 0.4 0.8 -3 -2 -1 -0 10 -8 ∆ F/C D (M -1 ) 104_C P (M)

A

B

Fig. 4.7. Binding isotherms for (A) the DNA/D system (pH = 7.0) and (B) DNA/ZnD (pH = 8.0) at different ionic strengths. λex = 385 nm, λem = 500 nm, T = 25°C, (□) I = 0.01 M,

(●) I = 0.10 M, (U) I = 1.0 M.

4.3.3 Dye displacement Assay

In order to better understand the nature of the binding process, a fluorescent intercalator displacement assay (Jenkins, 1997; Tse and Boger, 2004; Biver, Lombardi et al., 2006) has been applied to the DNA/D and DNA/ZnD systems by adding CT-DNA, previously saturated with the intercalating cyanine Cyan40, to samples of D or ZnD. The titrations show that binding mode 1 is still present, while the binding mode 2 is strongly reduced (Fig. 4.8A and B). This result shows that, the cyanine dye strongly interacts with DNA via intercalation, this hinders penetration of further agents between base pairs and suggests that D attempts to compete with Cyan40 for the intercalation sites (even if unsuccessfully). So

Dye I (M) 10-6 _{K1 (M}-1_{) 10}-4 _{K2 (M}-1₎ D 0.01 0.10 1.0 1.7 1.3 0.41 24 6.9 <1.0 [ZnD] 0.01 0.10 1.0 6.9 5.7 4.3 20 5.6 3.7 Proflavine 0.10 0.20 0.24 - - - 6.6 (a)] 2.0 (b) 4.3 (c)_(10°C)

binding mode 2 seems to be an intercalative one. Table 4.2 reports the values of

the ratio ∆φ2/∆φ1 of the amplitudes of binding mode 2 and binding mode 1, in the

absence and in the presence of the cyanine. For the DNA/D system the amplitude ratio between the two titration branches in the presence of cyanine is ten times smaller than that found with DNA alone, whereas for the DNA/ZnD system this ratio is about four times smaller. The higher amplitude found in the absence of the intercalator Cyan40 confirms that D binds to the intercalation sites.

0.0 0.5 1.0 1.5 2.0 -3 -2 -1 0 10 -8 ∆ F/ C D (M -1 ) 105C_P (M)

A

B

Fig. 4.8. (A) Fluorescence titration of D with DNA (U) and with DNA previously saturated with Cyan40 (●); I = 0.01 M, pH = 7.0, T = 25°C, λem = 500 nm. (B) Fluorescence titration of

ZnD with DNA (U) and with DNA previously saturated with Cyan40 (●); I = 0.01 M, pH = 8.0, T = 25°C, λem = 500 nm.

Table 4.2. The ratio between the amplitude of the binding mode 2 and the binding mode

1 in a traditional titration, ∆φ2/ ∆φ1, and with DNA previously saturated with Cyan40, (∆φ2/ ∆φ1)Cyan40. λem = 500 nm, T = 25°C.

4.3.4 Circular Dichroism measurements

The CD spectra of DNA/D and DNA/ZnD have been recorded under conditions where binding mode 2 is prevailing. The CD spectrum of the DNA/proflavine system has been recorded as well for comparison (Figure 4.9c). The CD spectra of DNA/D (Figure 4.9a) and DNA/ZnD (Figure 4.9b) are quite similar and their

∆φ2/ ∆φ1 (∆φ2/ ∆φ1)Cyan40

D/DNA -0.34 -0.03

shapes resemble that of proflavine intercalated into DNA (Figure 4.9c) with a large positive band appearing at longer wavelengths with respect to a negative small band (Dalgleish et al., 1969). The shift of (a) and (b)spectra, with respect to (c), along the wavelength axis is due to the position of the absorption band of proflavine, which is less energetic than the absorption bands of D and ZnD. Similarly, the higher amplitude of the proflavine band is to be ascribed to the higher absorptivity of this dye. The positive band, in proflavine derivatives, is characteristic of intercalation into DNA. Therefore, the observed CD behaviour confirms binding mode 2 should be interpreted in terms of intercalation.

350 400 450 500 -2 0 2 4 6 ∆ε λ (nm) c b a

Fig. 4.9. Circular dichroism spectra of DNA/D (a), DNA/ZnD (b) and DNA/proflavine (c) systems. CD = 3.0×10-5 M, CP = 3.0×10-5 M, I = 0.10 M (NaCl), T = 25°C. The similarity of

spectra (a) and (b) with spectrum (c) confirms the intercalative features of binding mode 2.

4.4

Supercoiled DNA unwinding

To obtain further information on the DNA binding mode, a DNA unwinding assay was performed by our collaborator at the University of Padova. Incubation of supercoiled DNA with Topoisomerase I gives rise to relaxed DNA and upon removal of the protein the relaxed state is maintained. On the contrary, when an intercalating agent is present in the reaction mixture, it alters the topological state of circular closed DNA and upon removal of both dye and enzyme, supercoiled DNA is suddenly regenerated. The magnitude of superhelicity is determined by the original amount of bound dye and the unwinding angle at the time of enzymatic religation (Zeman et al., 1998). An example of supercoiled DNA unwinding experiment is shown in Fig. 4.10. The presence of supercoiled plasmid is clearly evidenced starting from 1 µM of D or ZnD confirming their ability to

bind DNA through an intercalation mode. It is interesting to note that D and ZnD showed comparable efficiency in altering the enzyme-induced relaxation of the supercoiled plasmid suggesting comparable stability of the intercalated complexes.

Fig. 4.10. Topoisomerase I relaxation reactions of supercoiled pBR322 DNA performed in the presence of increasing concentrations of D and ZnD. S refers to supercoiled pBR322 DNA; C refers to relaxed pBR322 DNA obtained incubating the plasmid DNA with the enzyme in the absence of dye.

4.5

Kinetics of DNA/D and DNA/ZnD systems

The kinetic measurements with fluorescence detection have been carried out by means of a stopped-flow mixing unit using a blue laser diode (λ = 405 nm, 1 mW)

as a light source. Each experiment, performed under the conditions CP ≥ 10CD,

was repeated at least ten times, and the relaxation curves obtained were averaged by an accumulation procedure.

The kinetics of the interaction of D and its zinc complex with DNA were investigated in a range of dye and polymer concentrations where binding mode 2 is operative. In Fig. 4.11 is shown a typical relaxation curve for DNA/D system recorded at pH = 7.0. All kinetic traces were fitted by mono-exponential functions. In this case, the plot of the reciprocal time constant, 1/τ, as a function of DNA

concentration (CP) shows that the kinetic effect is independent of the

polynucleotide concentration (Figure 4.12A).

S C 0.1 0.25 0.5 1 2 5 C 0.1 0.25 0.5 1 2 5 µM

[ZnD]

0.0 0.2 0.4 0.6 0.8 0.7 0.8 0.9 1.0 Signal (V) Time (s)

Fig. 4.11. Stopped-flow relaxation curve for the DNA/D system monitored in the fluorescence mode. CD = 4.0×10-6 M, CP = 4.9×10-5 M, I = 0.10 M (NaCl), pH = 7.0, λex = 405 nm (laser), T = 25°C .

The DNA/ZnD system has been studied under analogous concentration conditions but at pH = 8.0. The analysis of the data reveals that also in this case the kinetics are independent of the DNA concentration (Figure 4.12B).

0.0 0.5 1.0 1.5 8 10 12 14 1/ τ (s -1 ) 104C_P (M) A 0.0 0.5 1.0 1.5 8 12 16 1/ τ (s -1 ) 104C_P (M)

B

Fig. 4.12. Dependence of the reciprocal relaxation time, 1/τ, on the polynucleotide

concentration for (A) DNA/D (pH = 7.0) and (B) DNA/ZnD (pH = 8.0) systems, I = 0.10 M (NaCl), pH = 7.0, λex = 405 nm (laser),T = 25°C.

The kinetic features of binding mode 2 could be rationalised on the basis of the reaction scheme (4.3)

(4.3)

D + S D,S k1 DS_int k_-1

where K0 is the equilibrium constant of the very fast step and D,S is a bound form

different from the final intercalate DSint. According to scheme (4.3) the kinetic

data could be fitted to eq. (4.4):

1 P 0 P 1 0 _k ) C K (1 C k K − + + = τ 1 (4.4)

As experimental data show that for both the investigated systems there is no dependence of the reciprocal relaxation time (1/τ) on the polynucleotide

concentration (CP), it can be concluded that K0CP>>1, and, thus, 1/τ = (k1 + k-1).

4.6

Discussion

Self-aggregation

Absorption spectra of acridine orange (Lamm and Neville, 1965) proflavine and other similar dyes (Chambers et al., 1974) display changes depending on dye concentrations which were ascribed to formation of dimers as principal species in equilibrium with the monomeric forms. Unfortunately, in the case of D and its Zn(II) complex, absorption spectra cannot be used to analyse the nature of the aggregate because, formation of this species takes place for concentrations greater than those suitable for absorbance measurements. However, NMR and potentiometric measurements (the latter not shown) confirm the presence of dimers at high concentrations of dye and provide some indications about the relative orientation of the adjacent molecules in these aggregates (Bazzicalupi et al., 2007).

A possible arrangement of the aggregate is depicted in Figure 4.13 showing a D dimer in which two face-to-face molecules are disposed in a head-to-tail mode. Similar considerations can be made for the Zn(II) complex of D, whose aggregation mode is deduced to be quite similar to that of the metal free ligand (Bazzicalupi et al., 2007). Analogous geometries of the stacked surfaces were also reported for proflavine (Obendorf et al., 1974; Neidle and Jones, 1975), acridine orange (Mattia et al., 1984; Bowler et al., 1989; Gordon et al., 2003) and acridine orange derivatives (Blears and Danyluk, 1967; Puliti and Mattia, 2001; Puliti et al., 2002).

N NH₂ NH2 N N H₂N H₂N N 4 3 2 1

Fig. 4.13. Proposed arrangement of D dimers.

In conclusion, it has been shown that the tendence of D and ZnD to self-aggregate is remarkably reduced, compared to proflavine (Fig. 4.2B). The cause of

this behaviour should be ascribed to the increased positive charges (H3D3+ and

ZnD2+_{) which enhance repulsion effects and possibly to steric effects due to the}

pendant arm that contrasts optimal overlap of the aromatic residues.

Dye binding to DNA

This study has shown that the bifunctional molecule D and its Zn(II) complex (ZnD) interact with double stranded DNA, forming stable complexes. Static measurements clearly show a biphasic behaviour for both systems, which reveals the occurrence of two different modes of binding.

The decrease of fluorescence emission and absorbance signals, observed for the first branch of titrations (binding mode 1), persists varying the ionic strength. The

values of K1 related to this mode of binding are two orders of magnitude higher

than those expected for intercalation reactions in spite of the fact that the

approximation made in eq. (4.2) ([S] = CP) would cause K1 to be somewhat

underestimated (Table 4.1). One could notice that the values of K1 for both

DNA/D and DNA/ZnD systems display a slight ionic strength dependence, possibly due to charge effects (Li et al., 2006). The major difference between the two systems lies in the fact that the affinity of ZnD for DNA is about four times greater than that of D, suggesting that in binding mode 1 the residue containing the zinc ion plays an important role. The observation that the first branch of the titration curves is not influenced by the presence of cyanine intercalated into DNA suggests that in binding mode 1 the interaction between dye and DNA occurs externally to the DNA cavities, with the polyamine residue possibly lying along the grooves of the nucleic acids.

Concerning binding mode 2, the enhancement of fluorescence emission and the observed red shift of the absorbance spectra during the second part of titrations

indicate base-dye interactions. The values of the related binding constant, K2, are

close to the value measured for the DNA/proflavine system (Table 4.1) which is known to bind double stranded nucleic acids by intercalation (Li and Crothers, 1969; Dourlent and Hogrel, 1976; Biver et al., 2003). Since D and ZnD bear +3

and +2 charges ([H3D]3+, [ZnD]2+), respectively, the slopes of LogK versus –Log[Na+]

plots could be much higher (≅ 3 and ≅ 2). The lower values obtained (0.7 for D and 0.4 for ZnD) suggest that not the entire molecule but only the proflavine residue takes part in binding mode 2. Actually the charge of this residue is +1 for D and smaller than 1 for ZnD. For these reasons we suggest that binding mode 2 corresponds to the intercalation of the aromatic residue into DNA, a result confirmed by CD experiments and unwinding of supercolied DNA.

The dye displacement assay shows for both systems that, upon the addition of DNA saturated with Cyan40, the first binding mode still remains whereas the second one is suppressed (Figure 4.8A and B). This behaviour indicates that D is not able to push Cyan40 out of the nucleotide cavities, otherwise a fluorescence increase would be observed at the wavelength of 500 nm where only the D/DNA complex, formed according to binding mode 2, is fluorescent. Since it is demonstrated that Cyan40 is intercalated between the base pairs of DNA (Biver, De Biasi et al., 2005), the binding mode 2 necessarily should correspond to an intercalative process. Similar considerations can be applied to the ZnD/DNA system.

The kinetics of D and ZnD binding to DNA according to binding mode 2 show that the values of the time constants of the two systems are similar, thus suggesting that the rate determining step involves a residue common to both dyes i.e. the proflavine residue. This is in agreement with the comparable binding constant observed in the presence/absence of the metal ion for the second equilibrium, which gives rise to comparable efficiency in DNA unwinding and similar CD behaviour.

The values of the rate constants are orders of magnitude lower than the value

expected for diffusion controlled reactions (Strehlow, 1992), this fact confirming

that the binding process, represented by the apparent reaction (4.1), is not a simple one but is composed of at least two steps. The kinetic features of binding mode 2 could, therefore, be rationalised on the basis of the reaction scheme (4.3).

The absence of concentration dependence of relaxation times indicates that D,S is

a stable complex (large value of K0) as the intercalate DSint.

We can conclude that this ligand and its Zn(II) complex bind calf thymus DNA forming complexes of considerable stability according to a biphasic behaviour defined by two different binding modes depending on the polymer to dye molar

ratio (CP/CD). At relatively low CP/CD values, both the metal-free ligand and its

Zn(II) complex associate with DNA externally to the base pairs, with principal

involvement of the polyamine moiety. On the other hand, at high CP/CD values

the interaction takes place via intercalation of the proflavine residue between base pairs, without significant effects deriving from the presence of Zn(II).