Amira 91

Glutathione (GSH) is a tripeptide with sequence ECG, where glutamic acid and cysteine are connected through a gamma peptide linkage. GSH can be oxidised to form glutathione disulfide (GSSG), where two glutathione molecules are connected through a disulfide bond. The structures of both of these compounds are shown in Figure 4.8 and denoted (1) and (2) respectively. Compound AH076 was initially reacted with both GSH and GSSG in order to compare observations made for the binding sites in insulin. Figures 4.9(a) and (b) show the mass spectra obtained for the reactions of AH076 with GSH and GSSG respectively.

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Figure 4.9: (a) reaction of glutathione (GSH) and AH076, with detected adducts containing ruthenium labelled i) – v), peaks labelled vi) - viii) are variations of complex AH076; * indicates an internal calibrant ion and _▲ indicates the isotope distributions of two of the adducts; (b) mass spectrum of the reaction between oxidised glutathione (GSSG) and AH076

As can be seen in Figure 4.9(a), on reaction of GSH with AH076, a number of ruthenium-containing adducts were detected, and these are summarised in Table 4.2.

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Table 4.2: Ruthenium(II) adducts detected after reaction with glutathione (GSH) Observed GSH–Ru adduct m/z Assignment

(i) 718.12675 [GSH + {(bip)Ru(bipy)} – H]+ (ii) 562.05797 [GSH + (bip)Ru} – H]+ (iii) 540.08508 [Glu + {(bip)Ru(bipy)} – H• - H]+ (iv) 489.01624

[Glu + Cys + Ru(bipy) – H• – H]+ (v) 457.04823

[CH2O2 + {(bip)Ru(bipy)} – H]+

The adducts detected indicate, as was observed with angiotensin(II) and bombesin, that AH076 loses its ligands in different combinations. The major product detected was that at m/z 457.05 where the {(bip)Ru(bipy)} is bound to CH2O2 through loss of the chloride ligand. This is more likely to originate from the glutamic acid side chain than the C-terminus of the peptide, as a second adduct at m/z 540.08 was assigned as {(bip)Ru(bipy)} plus glutamic acid as a whole. This supports the observations of binding to glutamic acid, and potentially tyrosine, in insulin showing that in addition to nitrogen, ruthenium(II) has an affinity for –OH groups, with

binding occurring through loss of H•. AH076 was also detected as Ru(bipy) bound

to both glutamic acid and cysteine, indicating that with loss of the arene, ruthenium(II) is able to be coordinated by both amino acids.

Figure 4.8(B) shows the mass spectrum obtained after reaction between AH076 and oxidised glutathione (GSSG). This time, the sulfur atom of cysteine is in a disulfide bond, and so ruthenium(II) is unable to bind to the cysteine. The peak at m/z 613.16 corresponds to the unreacted oxidised glutathione, showing that the

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majority is unable to react with the ruthenium(II). The major adduct observed is the same as for GSH, {(bip)Ru(bipy)} plus CH2O2; a peak at m/z 484.12 was also observed which corresponds to GSSG minus glutamic acid, supporting the conclusion that the CH2O2 originates from glutamic acid and not the C-terminus of the peptide.

In order to determine if AH076 could bind to cysteine when it is oxidised further to sulfonic acid, as was first thought with the oxidised insulin B-chain, glutathione was reacted with hydrogen peroxide in order to oxidise the cysteine side

chain to sulfonic acid (Cys-SO3H). The structure of this compound, (3), is shown in

Figure 4.10 with the corresponding peaks detected in the mass spectra, showing the conversion of cysteine to the fully oxidised form.

Figure 4.10: Oxidation of cysteine in GSH (1) to cysteine sulfonic acid (3); peaks at

m/z 308.09 and 356.08 show the increase in 48 Da through addition of three oxygen

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CAD was performed on glutathione both before and after reaction with H2O2 and determined that both the y2 and b2 fragments increase by 48 Da, showing the addition of three oxygen atoms to the sulfur of cysteine, forming cysteine sulfonic acid. Figure 4.11 shows the mass spectrum obtained after reaction between GSH (3) and AH076.

Figure 4.11: Mass spectrum of oxidised GSH (3) reacted with AH076; inset shows adduct at m/z 611.02 with ▲ indicating its isotope distribution as compared to the simulation. * indicates an internal calibrant ion

As before, the major adduct detected is the addition of CH2O2 to {(bip)Ru(bipy)}; however, a minor product was also observed at m/z 611.03, and was assigned as

GSH (3) plus {Ru(bipy)} with a 2H• loss. Potentially, as a minor pathway, binding

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as from the –OH group of glutamic acid. This also indicates that ruthenium(II) is not binding to the oxidised cysteine through displacement of –OH, as was initially thought in the reaction with the oxidised insulin B-chain.

4.4 Conclusion

The products from reactions of two organometallic ruthenium(II) arene anticancer complexes has been investigated using FTICR mass spectrometry with the peptides angiotensin(II), bombesin, and glutathione, as well as insulin and the oxidised insulin B-chain. In particular, insights into ruthenium(II) binding sites have been gained using collision activated dissociation (CAD) and electron capture dissociation (ECD) to fragment the products. The amino acids primarily involved in coordination to ruthenium(II) have been identified as histidine, and methionine, with additional binding to arginine, phenylalanine, glutamic acid and, potentially, the nitrogen atoms of the backbone amides. Two different binding modes were assessed; at low ratios of ruthenium(II) to peptide, the ruthenium(II) compounds were observed to lose both the arene and the chloride ligands, providing the ruthenium with four possible binding sites. At higher ruthenium(II) to peptide ratios, an additional species was observed in which ruthenium(II) lost only its chloride ligand. Interestingly, ruthenium(II) appeared to preferentially coordinate to the phenylalanine residue of angiotensin(II) and not histidine as for the lower ratios; an observation that has been made for the first time. There was no evidence for the disruption of the disulfide bonds of insulin after reaction with ruthenium(II). Instead, the possible Ru(bipy) binding sites include histidine, glutamic acid, and tyrosine, which is in contrast to data obtained for cisplatin. Binding to His10 was further supported by reaction with the oxidised insulin B-chain, and binding to glutamic acid was investigated using

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glutathione and its oxidised form, GSSG. The major product observed for both GSH

and GSSG involved the addition of CH2O2 to {(bip)Ru(bipy)} indicating binding of

ruthenium(II) to glutamic acid can occur. The use of high mass accuracy has been demonstrated in order to make peak assignments, and subsequently identify the binding sites of ruthenium(II) complexes, with a high degree of confidence.

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Chapter 5: Further Applications of