Chapter 4 Effect of varying the number of pendant groups on DNA binding
4.2.1 DNA binding studies performed using ESI mass spectrometry
ESI-MS was first used to compare the affinity of nickel complexes with different numbers of pendant groups towards the tetramolecular G-quadruplex Q4, unimolecular G-quadruplex Q1 and dsDNA D2. This technique has been previously used to obtain information about the number, stoichiometry and relative amounts of non-covalent adducts formed between metal complexes and G-quadruplex or dsDNA.[120, 127, 141, 222, 223] Stock solutions of Q1 and Q4 used in these ESI- MS experiments were annealed under conditions which ensured that they were present in parallel conformations (Chapter 2.3.3). Figure 4.2 shows the negative ion ESI mass spectra of free Q4, and solutions containing a 3:1 ratio of nickel Schiff base complexes and Q4. Each spectrum shows ions of medium or high abundance at m/z 1675.2 and 2010.4, which are assigned to [Q4 + 4NH4 – 10H]6- and [Q4 + 4NH4 – 9H]5-, respectively. These ions arise from Q4 molecules with a total of four ammonium ions bound, as expected for a quadruplex structure consisting of five tetrads and one monovalent cation located in between each tetrad.[127]
The results of the mass spectral study indicate that the affinity of the new nickel Schiff base complexes towards Q4 generally increased with the number of pendant groups. In the case of complex (87), which contains only one pendant group, the mass spectrum (Figure 4.2 (c)) only showed ions from free Q4. In contrast, when the nickel complex contained either two or three pendant groups (complexes (71) and (77), respectively), ions from non-covalent complexes consisting of one nickel molecule bound to Q4 were also present in low to medium abundance (Figures 4.2 (d) and (e)).
149 Figure 4.2 Negative ion ESI mass spectra of solutions containing free Q4 or different nickel Schiff base complexes and Q4 at a 3:1 ratio. (a) free Q4; (b) Q4 + (54); (c) Q4 + (87);
(d) Q4 + (71); (e) Q4 + (77) and (f) Q4 + (89). = free Q4; = {Q4 + (Ni)}; = {Q4 + 2(Ni)}.
In the case of (71), these ions were at m/z 1797.2 and 2156.8, and are assigned to [Q4 + (71) + 4NH4 – 12H]6- and [Q4 + (71) + 4NH4 – 11H]5-, respectively.
150 When the nickel complex used was (77), the corresponding ions were observed at m/z 1820.5 and 2184.8. The above ions and adduct(s) will hereafter be referred to collectively as {Q4 + (71)} and {Q4 + (77)}, and this terminology will be used in future to describe non-covalent adducts consisting of one or more of the other novel nickel Schiff base complexes and different DNA molecules. Complex (89), with four pendant groups, exhibited the highest affinity towards Q4. This is supported by Figure 4.2 (f), which shows no ions from free Q4, but does contain ions of high, and low to medium abundance, from {Q4 + (89)} and {Q4 + 2 (89)}, respectively. The prevalence of ions from {Q4 + (89)} in the Figure 4.2 (f) supports the hypothesis that Q4 may have one high affinity binding site for this nickel complex. In contrast, Figure 4.2 (b) shows that under the same conditions the literature complex (54) formed non- covalent complexes consisting of one or two nickel molecules bound to Q4 in roughly equal amounts. This suggests that Q4 has two binding sites for (54) with similar affinities.
Analogous DNA binding experiments were conducted by ESI-MS using the parallel unimolecular G-quadruplex Q1 and dsDNA D2. Figure 4.3 shows how the relative abundances of ions varied in ESI mass spectra of solutions containing a 3:1 ratio of one of the new nickel complexes, or (54), and Q1, Q4 or D2. Relative abundances were obtained by adding the abundances of all ions arising from either free DNA or non-covalent adducts containing a specific number of bound nickel molecules, and dividing by the total abundance of all ions in the spectrum, and converting the results to percentage values.[128] The results obtained show that the nickel complex with one pendant group, (87), did not form non-covalent ions with any of the three types of DNA investigated. When the [Ni]:[DNA] ratio was increased to 6:1, the resulting mass spectra still did not show any ions from {Q1 + (87)} or {D2 +
151 (87)}, whilst the relative abundances of ions from {Q4 + (87)} was very low (Figure S4.1). This confirms that (87) has a very low affinity towards DNA in general, and suggests that the low overall charge of the complex may limit its DNA binding capability.
Figure 4.3 Relative abundances of ions in ESI mass spectra of solutions containing a 3:1 ratio of nickel Schiff base complexes and dsDNA (D2), unimolecular qDNA (Q1) or tetramolecular qDNA (Q4): (a) solutions containing (54); (b) solutions containing (87);
(c) solutions containing (71); (d) solutions containing (77) and (e) solutions containing (89).
Complex (71), which contains two pendant groups, was shown previously to interact with Q4. In contrast, Figure 4.3 (c) shows that mass spectra of solutions containing this nickel complex and either Q1 or D2 did not contain ions of significant abundance from non-covalent complexes. Complex (77), with three pendant groups, also did not show an ability to form non-covalent complexes with D2, but unlike (71) did produce ions of low abundance from {Q1 + (77)}. In contrast to all of the above nickel complexes, (89) did form significant amounts of non-covalent complexes with
152 Q1, whilst still exhibiting a relatively low degree of affinity towards D2. This observation suggests that the number of pendant groups in these metal complexes can have a major effect on their ability to bind to G-quadruplexes.
Inspection of Figure 4.3 shows that increasing the number of pendant groups in the new class of nickel Schiff base complexes did not greatly increase their ability to interact with the dsDNA D2. In contrast, the literature complex (54) did show evidence of binding to D2, which was more notable in solutions containing a 6:1 ratio of [Ni]:[DNA] (Figure S4.1 (a)). It is also noteworthy that (89), with four pendant groups, did not bind as strongly to D2 as (54). This conclusion is supported by the greater percentage of ions from free DNA in the spectrum containing the former nickel complex (Figure 4.3 (e)), and is also evident in the data obtained from spectra of solutions containing a higher [Ni]:[DNA] ratio (Figure S4.1 (e)). Figure 4.3 also shows that the abundances of ions from free DNA was lower in the case of spectra containing (89) and either Q1 or Q4, than for the corresponding spectra of solutions with (54) present. Taken together, the results presented above suggest that the affinity of (89) towards Q1 and Q4, and its selectivity for these DNA molecules over D2, is at least comparable to, if not greater than that exhibited by (54).