SECCIÓN UNICA De las obligaciones solidarias

complement our previously published work in which the addition of chloride salts to solutions containing pyridylpolyamine anthracene 5 (Scheme 4.1) were shown to produce an increase in anthracene-sensitized DNA photocleavage 12. Reactions were conducted in the presence and absence of 150 mM NaCl and 260 mM KCl in order to approximate the conditions of high ionic strength that exist in the cell nucleus 13-16. UsingUV-visible spectrophotometry in combination with circular dichroism, fluorimetry, and thermal melting data, we demonstrated that the

photocleavage increase was accompanied by a salt-induced change in DNA binding mode from intercalation to external interactions 12. In photocleavage reactions containing the ROS

scavengers sodium benzoate, D-mannitol, and sodium azide and deuterium oxide, which

increases the lifetime of singlet oxygen, we then showed that the addition of the salts triggered a sharp increase in the pyridylpolyamine anthracene-sensitized production of DNA damaging hydroxyl radicals and singlet oxygen 12. Taken together, the previously published data point to a close relationship between DNA photocleavage, reactive oxygen species, and anthracene binding mode. The microenvironment of the DNA intercalation pocket can prevent chromophores from freely interacting with ground state triplet oxygen 8, 48. When bound externally 8, 12 or in the DNA minor groove 49 however, anthracene and other chromophores are considerably more accessible to oxygen and have the potential to generate DNA damaging reactive oxygen species more efficiently.

The present work examines the effects of 150 mM NaCl and 260 mM KCl on DNA photocleavage by bis-anthracene dye 2 and its monomeric analog 4. Several major conclusions can be drawn from this study. Under conditions of low ionic strength, the absorption, viscosity, CD, and thermal melting data presented in this paper indicate that bis-anthracene 2 engages in persistent intercalative binding (Figs. 4.2 through 4.7). In the case of 4 however, the DNA interactions were influenced by the molar ratio of anthracene to DNA bp (r). The viscosity and absorption data pointed to intercalation at r values less than 0.27 (Figs. 4.3 and 4.4A). At higher molar ratios, the absorption spectra of mono-anthracene 4, taken together with CD and thermal melting data, suggest that levels of intercalation are decreased and that minor groove interactions predominate (Figs. 4.2C, 4.3C, and 4.4A). The concept of multiple anthracene binding modes was first considered by Rodger and co-workers, who employed circular and linear dichroism to

study the interactions between an anthracene-9-carbonyl derivative of spermine and the polynucleotides [poly(dA-dT)]2 (AT) and [poly(dG-dC)]2 (GC) 44, 50. At low r values,

intercalation was the dominant binding mode, but at high r values, groove-bound anthracene was detected. In the case of GC, intercalation was favored, and groove binding only occurred when the intercalative mode was saturated. In the case of AT however, the intercalative and groove binding modes were competitive 50. CD data suggested that groove binding might involve anthracene-anthracene stacking interactions 44. In the UV-visible absorption titrations shown in Fig. 4.2, CT DNA was added to fixed concentrations of anthracene. At initial r values, vibronic bands of mono-anthracene 4 display significant hypochromicity in the absence of red shifting, suggesting that groove binding interactions precede intercalation under the conditions of the titration experiment (Figs. 4.2C and 4.3C).

Additional important conclusions drawn from the present study involve the relationship between DNA interactions and ionic strength. The absorption, viscosity, CD, and thermal melting experiments reported in this paper indicate that the addition of 150 mM NaCl in combination with 260 mM of KCl promotes a transition from intercalation to minor groove binding at high bis-anthracene 2 to DNA bp molar ratios (r ≥ 0.17) and mono-anthracene 4

molar ratios (r ≥ 0.039). Accordingly, Tuite and Nordén have shown that the predominant

interaction between the phenothiazinium dye methylene blue and [poly(dA-dT)]2 (AT) changes from intercalation under conditions of low ionic strength to groove binding with increasing salt concentrations 51. Using CT DNA, Kumar and co-workers demonstrated that anthracene

derivatives substituted at the 10 and/or 9 position exhibit a salt-induced change in binding mode similar to methylene blue 10, 31-32. Groove binding compounds interact with DNA by making close van der Waals contacts with the walls of the minor groove. Van der Waals interactions are

short-range forces that fall off exponentially as a function of increasing intermolecular distance. As previously mentioned, conditions of high ionic strength decrease the width of the DNA minor groove 22. It is conceivable that this salt-induced structural change promotes a transition from intercalation to groove binding by enabling anthracenes 2 and 4 toachieve optimal van der Waals contact distances 31. In lieu of groove binding, we detected external interactions in our published work with compound 5 (Scheme 4.1). This was perhaps due to unfavorable steric interactions between the DNA minor groove and the relatively bulky pyridylpolyamine side chain of the anthracene 12. While the energies associated with individual van der Waals contacts are very small, even at short intermolecular distances, the large number of contacts within the DNA minor groove makes total van der Waals interactions a major intermolecular force. This points to the importance of shape and size complementarity in the design of minor groove binding ligands 52-53.

The UV-visible absorption titrations in Fig. 4.2 and the CD spectra in Fig. 4.5 suggest that the combination of chloride salts, in addition to changing binding mode, reduces the overall affinity of anthracenes 2 and 4 for DNA. The latter result was expected for the following reasons. When present in high concentrations, sodium(I) and potassium(I) cations reduce the binding of positively charged ligands by effectively competing for negatively charged sites on the DNA 18. Also, at high concentrations the cations promote duplex unwinding, condensation, and other structural changes that reduce ligand affinity 10, 19-20. Although the chloride salts lowered overall binding, anthracenes 2 and 4 were affected to different extents. Under conditions of low ionic strength, the UV-visible titration and photocleavage data suggest that 2 and 4 have similar affinities for DNA (Figs. 4.2A and 4.2C). Bis-anthracene 2 and mono-anthracene 4 each reached saturation at approximately the same CT DNA concentration (297 µM bp) and produced similar

levels of photocleavage (Figs. 4.1 and 4.S1). However, in the presence of 150 mM NaCl and 260 mM KCl, bis-anthracene 2 appeared to have significantly more DNA affinity than 4, reaching saturation at 645 µM bp CT DNA compared to 2888 µM bp for the mono-anthracene and producing more photocleavage at most of the dye concentrations tested (Lanes 8 to 12 in Fig. 4.1; Lane 6 in Fig. 4.S1). As a result, the addition of the chloride salts increased bis-anthracene 2

photocleavage over a broader range of r values compared to mono-anthracene 4. This reduced the lower limit for observing a salt-induced photocleavage enhancement from 2.5 µM for the mono-anthracene dye to 0.25 µM for bis-anthracene 2 (Figs. 4.1 and 4.S1; 350 nm, pH 7.0, 22 °C).

4.3.8. Concluding Remarks. Anthracenes and their derivatives have a number of current