POSICIONAMIENTO DEL SENSOR LÁSER

The presence of the N-H protons on the ethylenediamine ligand has been shown to be essential for the anticancer activity of ruthenium-arene anticancer complexes.68Their role is to stabilise, by H-bonding, the binding of nucleobases to ruthenium as is shown in Figure 1.10. Similar observations have been made with cisplatin derivatives, in these studies removal of am(m)ino protons reduces their cytotoxic activity.97The importance of H-bonding between the exocyclic

interconvert in order to bind to guanine bases.70The mechanism for this interconversion is unknown and to date there have been no investigations into mechanisms for interconversion of chiral amine ligands on metal centres. Chapter 3 of this thesis investigates the amino proton exchange rates of1 ([Ru(η6-bip)(Et- en)Cl]+) in aqueous solutions. The data are then used to infer the possible

mechanism for the interconversion of the stereocentres. The author believes this is the first such in-depth study into a widely observed phenomenon in six co-

ordinate metal complexes. Further analysis in Chapter 4 investigates the effects of electron donation to the metal from other ligands on the exchange rates of the amino protons. Together both studies show evidence for a complicated interconversion mechanism. It is believed that the determination of this

mechanism will allow for further understanding of ligand choice in the design of ruthenium(II) anticancer complexes. Further to this, suggestions are made as to how to control this interconversion and highlight the difficulties involved in attempting to separate stereoisomers of this type are discussed. The discovery of the influence of other ligands on the mechanism will provide a greater

understanding of the exchange pathway and will have implications not only in drug discovery but also in the development of asymmetric-catalysts.

Chapter 5 investigates the shape changes that can occur for short strands of oligonucleotides (DNA). Previous work has shown that ruthenium(II) arene

complexes can cause unwinding of supercoiled plasmid DNA.98,99Unlike cisplatin, there is currently no crystal structure of ruthenium bound DNA.100As a result any kinking, stretching or bending of the DNA by ruthenation has not been

characterised by x-ray crystallography and so other methods must be employed. The author investigated the use of Ion-mobility Mass spectrometry (IM-MS) and

Förster Resonance Energy Transfer spectroscopy (FRET) for determining shape changes to DNA strands as a result of ruthenation. Understanding DNA shape changes will provide a greater insight into the mechanism of action for this class of compounds. Such knowledge might allow new complexes to be developed which will selectively change the shape of DNA to give rise to the most cytotoxic lesion on DNA, and hence the greatest anticancer activity.

1.9 Aims

The aims of the work described in this thesis are listed below.

1) To investigate the mechanism of stereochemical inversion between (R*,S*) and (S*,S*) isomers of [Ru(η6-bip)(Et-en)Cl]+(c.f.: Fig. 1.13). The question as to whether inversions occur on the chiral nitrogen or the chiral ruthenium or both in the complex in aqueous solutions is studied by

observing the exchange rate of amino protons in [Ru(η6

-bip)(en)Cl]+ at various pH values, temperatures and ionic strengths using nuclear magnetic resonance spectroscopy (NMR). Knowledge of this mechanism of exchange is important since the use of racemic mixtures is usually avoided in pharmaceuticals. The ability to control this exchange may allow the formation of a racemic mixture to be avoided.

2) To investigate the effects of nitrogen based π-acceptor ligands (pyridine

derivatives) in the Z position of [Ru(η6

-bip)(en)Z]n+ on the rate and mechanism of exchange using NMR and computational methods. A comparison of the exchange rates using a Hammett plot provided an understanding of the electronic effects of the monodentate ligand on the exchange rates. A combination of the data in Chapters 3 and 4 will provide a greater understanding of the effects of ligands on the ability of a complex to undergo inversion. The data will also assist the design of improved anticancer complexes and reduce the complications arising from stereoisomers interconverting in aqueous solutions.

3) To use Ion-Mobility Mass Spectrometry (IM-MS) to investigate changes in the 3D conformation of the short DNA strand, d(CACGTG), as a result

of interaction with {Ru(η6

-bip)(en)}2+. This should provide an understanding of the conformational changes to DNA molecules induced by binding to ruthenium-arene complexes. This appears to be the first IM- MS study of ruthenated DNA.

4) To use IM-MS to determine the collision cross sections of ruthenium-arene complexes. This appears to be the first usage of IM-MS to determine the collision cross section of a small molecule (< 100 Å2). To facilitate this a new calibration process was developed. Previously this technique had only been suitable to larger molecules.

5) To investigate the potential of Förster Resonance Energy Transfer (FRET) to determine shape changes to 30mer oligonucleotides induced by binding to metal-based anticancer drugs. It was hoped that these results would allow analysis bending, kinking or any other shape changes on longer strands of DNA. Initial studies were carried using cisplatin with the intention of developing the technique for use with ruthenium-arene anticancer complexes.