Bis-substituted considerations

As mentioned previously, the major aim of this Chapter is to study what bis-substituted complexes are formed and, perhaps more importantly, which combinations of nucleobases are precluded, depending on the steric environment. This bis-substitution discrimination may inform further drug design studies, due to the mechanism of action of similar chemotherapy agents as discussed in section 4.1.2 above. In the following discussion, bbme and bbmp will be examined first, with appropriate conclusions drawn regarding the possible behaviour of the bbmm system following.

The bbme BE results are presented below, in Table 4.10, alongside the ESI-MS results first presented in section 4.3.1.2.2 for ease of comparison. These BE values were calculated as per Eq. 4.1 in section 4.2.3 above and are presented in kcal/mol. All BE values displayed in the following tables are for the most energetically favourable conformer, given that various possible orientations of the nucleobases around the palladium atom are possible (i.e. head-to-head, etc.). Further, where the complexes contain two different nucleobases, the BE

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value presented indicates the lowest energy combination and are presented this way in Table 4.10. For example, the first entry indicates that the [Pd(bbme)(3- methyladenine)(Inosine)]+ complex has a BE of –57.14 kcal/mol when an inosine moiety binds to a [Pd(bbme)(3-methyladenine)(H2O)]+ mono-substituted

complex.

Table 4.10 –Summary of calculated BE values for the most energetically favourable bis-functional adducts of the bbme system, as compared to the equivalent ESI-MS results. All values are in kcal/mol.

ESI-MS Result (% abundance)

Complex BE Inos.- Cyt. Inos.-3- MeA All three nuc. Pd(bbme)(3-methyladenine)(Inosine) -57.14 - 10.4 12.5 Pd(bbme)(Cytidine)(Inosine) -51.51 8.7 - 9.5 Pd(bbme)(Inosine)2 -49.88 9.8 7.2 2.5 Pd(bbme)(Cytidine)(3-methyladenine) -41.08 - - 0 Pd(bbme)(Cytidine)2 -27.02 0 - 0 Pd(bbme)(3-methyladenine)2 +14.74 - 0 0

The first aspect that is immediately noticed is the relatively greater magnitude in calculated BE values compared to the mono-substituted complexes. This is not unexpected, as the nucleobases themselves provide some steric hindrance, leading to consistently larger binding energies. Also noticeable is the large range of BE values, indicative of the large effect wielded by the sterically active carrier ligand in combination with the nucleobases.

Generally, as can be seen in Table 4.10 above, the computationally derived BE values follow a very similar trend to that observed in the ESI-MS results, with the possible exception of the inosine-cytidine competition experiment. This may be due to the fact that the two complexes are energetically similar, according to the BE values, and as such may be sensitive to experimental stoichiometric ratios. Certainly, where more competition is present, as seen in the final experiment with all three nucleobases, the trend of the experimental results matches the calculated BE values.

Perhaps more interestingly is the absence of three bis-complexes; namely the cytidine-3-methyladenine, bis-cytidine and bis-3-methyladenine adducts. The pleasing aspect of this non-appearance of these adducts in the experimental data is that these particular complexes registered the largest BE values, showing that, in this particular example, the semi-empirical PM3 method is capable of not only describing the trends of observed species, but is able to identify unobserved species. This discrimination between observed and absent species (in the ESI-MS data) appears to lie somewhere between approximately –50 kcal/mol and –41 kcal/mol (in the computational data). This range is only approximate, given the limitations of the PM3 method and is not designed to form the basis of a steadfast rule; it is merely an observation at this point.

The bbmp results (Table 4.11) display a similar trend to the bbme results, reinforcing the similarity of these ligands. However, closer inspection reveals subtle differences between the bbme system discussed above and the bbmp system described below.

Table 4.11 – Summary of calculated BE values for the most energetically favourable bis-functional adducts of the bbmp system, as compared to the equivalent ESI-MS results. All values are in kcal/mol.

ESI-MS Results Complex BE Inos.- Cyt. Inos.-3- MeA All three nuc. Pd(bbmp)(3-methyladenine)(Inosine) -57.61 - 22.6 12.0 Pd(bbmp)(Cytidine)(Inosine) -54.21 18.3 - 10.9 Pd(bbmp)(Inosine)2 -52.69 14.8 21.8 7.8 Pd(bbmp)(Cytidine)(3-methyladenine) -45.02 - - 0 Pd(bbmp)(Cytidine)2 -23.60 6.3 - 0 Pd(bbmp)(3-methyladenine)2 +15.14 - 0 0

Unlike the bbme system above, the trends seen in the ESI-MS for all three experiments with the bbmp ligand match the trends seen in the equivalent BE results, with respect to those species observed, as well as those species which are not observed. This is again a pleasing result that supports the use of PM3 as a possible screening method in the development of cytotoxic agents.

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However, what must be noted here is the ambiguity of the bis-cytidine results. According the BE results, which explain all other experimental observations to this point, the bis-cytidine adduct should not form at all, especially given the absence of a cytidine-3-methyladenine complex, which appears to be more energetically favourable. This may again be a stoichiometric artefact from the experimental procedure, especially as this particular complex is not seen in the experiment involving a greater level of competition.

Where competition for the second binding site is greater, energetic considerations would gain more importance. This is consistent with the BE results for the experiment involving all three nucleobases, with the same three adducts precluded as seen in the bbme system. Again, the PM3 results suggest a theoretical cut-off energy whereby that particular combination is less likely to be observed in the ESI-MS results, of somewhere between –53 and – 45 kcal/mol. To cautiously expand on this theme, keeping in mind the energetic limitations of semi-empirical methods, it would appear that this energy cut-off may lie between –50 and –45 kcal/mol if the results for both ligand systems are combined (and the bis-cytidine positive result is discounted).

Whilst, due to unforseen circumstances, the ESI-MS results involving the bbmm carrier ligand did not work as planned, some informed comment can still be made. The BE values for the bbmm ligand in contained in Table 4.12 below.

Table 4.12 – Summary of calculated BE values for all possible bis-functional adducts of Pd-bbmm. All values are in kcal/mol.

Complex BE Pd(bbmp)(3-methyladenine)(Inosine) -58.98 Pd(bbmp)(Inosine)2 -50.58 Pd(bbmp)(Cytidine)(Inosine) -43.80 Pd(bbmp)(3-methyladenine)(Cytidine) -36.99 Pd(bbmp)(Cytidine)2 -14.68 Pd(bbmp)(3-methyladenine)2 +12.49

Using the results for the two previous studies as a guideline for the interpretation of the BE values above, it would be expected that the more sterically-hindered system, bbmm, would only form the 3-methyladenine-inosine and bis-inosine adducts, with all other nucleobase combinations precluded, including the cytidine-inosine adduct that was identified in all other experiments where this combination was possible. This conclusion is based on the ESI-MS results and the subsequent PM3 BE values obtained for the complexes around the threshold of inclusion and preclusion seen in the two other ligand systems. Whether this prediction holds true can only be realised through further experimental investigation.

Table 4.13 –Summary of calculated BE values for all possible bis-functional adducts of Pd-bbmm. All values are in kcal/mol.

Nucleobase to be added

Complex Inosine Cytidine 3-methyladenine

Pd(bbmm)(Inosine) 19.38 27.60 13.34

Pd(bbmm)(Cytidine) 16.72 33.76 12.36

Pd(bbmm)(3-MeA) 10.98 20.88 12.50

4.3.3 Summary

Evidence presented here shows that ESI-MS can be used in order to determine site selectivity for platinum(II) and palladium(II) complexes that possess a variety of steric influences on the binding site. This nucleoconstituent selectivity, as well as the vagaries of the “di-aqua” species, have been fully characterised using semi-empirical methods, providing insights into all aspects of the experimental procedure. Further, this study shows that a semi-empirical PM3 methodology may be used to prudently design similar carrier ligands of a more targeted nature, as the systems studied here agreed with the general trends seen in the ESI-MS data.

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Scope for further work in this area is extensive, as a variety of carrier ligands may be synthesised, for both platinum(II) and palladium(II), with a variety of influences over the coordination sphere, steric or otherwise, , lead by PM3 calculations, as these are computationally cheap and generally yield good qualitative information. Further competition experiments using ESI-MS can also be envisioned, where short oligonucleotide sequences may be used in lieu of the nucleoconstituents, which would provide additional information on the specific binding patterns of the bis-functional adducts of these sterically restricted complexes, as oligonucleotide sequences would have their own unique restrictions.