Formation of glutathione sulfenate and sulfinate complexes by an organoiridium(III) anticancer complex

Abstract

The organoiridium(III) anticancer complex [(η⁵-Cp^xbiph)Ir(phpy)py] (Cp^xbiph = biphenyltetramethylcyclopentadienyl, phpy = phenylpyridine, py = pyridine) unexpectedly reacts with the tripeptide glutathione in the presence of coenzyme NADH to give glutathione sulfenate (Ir–S(O)G) and sulfinate (Ir–S(O)₂G) complexes. These new adducts may play a role in the anticancer activity of such organoiridium complexes.

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The platinum drug cis-[PtCl₂(NH₃)₂] (cisplatin) and the related complexes carboplatin and oxaliplatin have made a major contribution to cancer chemotherapy in the last 30 years.¹ Most attention in relation to their mechanism of action has focussed on DNA as the target.² Recently interest in platinum binding to peptides and proteins has emerged in order to understand not only off-target effects which cause toxicity, but also platinum transport.³ The tripeptide GSH (γ-L-Glu-L-Cys-Gly) is highly abundant in cells and is thought to be involved in platinum detoxification.⁴ In common with many other transition metal ions, Pt^II, a ‘soft’ metal ion, binds strongly to thiolate sulphur, a ‘soft’ ligand. GSH is an important antioxidant, preventing damage to cellular components caused by reactive oxygen species.⁵ Thus, reactions of metal complexes with GSH can affect the redox state of cells.⁶

We have previously reported that, unlike platinum drugs, some half-sandwich Ru^II arene anticancer complexes form glutathione complexes that rapidly undergo oxidation at the bound thiolate sulfur to give unusual sulfenato and sulfinato adducts.⁷ Curiously this appears to promote subsequent reactions with DNA. The sulfenato ligand can be displaced by the DNA base guanine.⁸ Oxidation of bound thiolate sulfur can also occur when the cysteine thiolate is in a protein (e.g. albumin, glutathione transferase) although the local reactivity of cysteine residues depends on their environment and the nature of the arene.⁹ This poses the question as to whether thiol-mediated redox reactions could play a role in the mechanism of action of other half-sandwich transition metal complexes.

Organometallic cyclopentadienyl Ir^III complexes exhibit promising anticancer activity.¹⁰ We have reported previously the formation of the Ir-GSH adduct [(η⁵-Cp^xbiph)Ir(phpy)(SG)]⁻ (Ir–SG, Cp^xbiph = biphenyltetramethylcyclopentadienyl, phpy = C^N-chelated phenylpyridine ligand), Chart 1, by substitution of the chlorido and pyridine (py) ligands in the anticancer complexes [(η⁵-Cp^xbiph)Ir(phpy)Cl] (Ir–Cl)¹¹ and [(η⁵-Cp^xbiph)Ir(phpy)(py)]⁺ (Ir–py) with glutathione, reactions which may influence their potency.¹² In the work reported here we have investigated whether Ir–SG is unreactive, as might be expected for a third row transition metal with a sulfur ligand, which might mean that this adduct would be a ‘dead-end’ product in cells with little involvement in anticancer activity, or perhaps can undergo oxidation at sulfur in a similar manner to Ru^II arene complexes.

Chart 1 Iridium complexes studied in this work.

First we investigated the formation and stability of the iridium glutathione adducts. To a solution of complex Ir–py, 3.5 mol equiv. GSH was added. After 12 h, 95% of Ir–py had reacted with formation of complex [(η⁵-Cp^xbiph)Ir(phpy)(SG)]⁻ (Ir–SG).¹² There was little change to the solution after further 18 h. When the above reaction was studied in the presence of 104 mM NaCl (mimicking the chloride concentration in blood plasma), 90% of the {Ir(η⁵-Cp^xbiph)} fragment (which is readily monitored by the sharp ¹H NMR singlets for the Cp methyl groups between 1.5 and 2.0 ppm) was still in the form of Ir–SG and only 10% was present as Ir–Cl or Ir–py after 17 h (Fig. S1†), indicating the high stability of Ir–SG.

However, when coenzyme NADH (2 mol equiv.) was added to a solution containing 95% Ir–SG and 5% Ir–py, surprisingly oxidation of NADH to NAD⁺ was observed (Fig. 1, Table 1, Reaction 1). Notably this led to ca. 34% of Ir–SG being oxidized to the sulfenato complex [(η⁵-Cp^xbiph)Ir(phpy)(S(O)G]⁻ (Ir–S(O)G). Complex Ir–S(O)G gave two sets of peaks for the Cp^xbiph methyl protons at 1.93, 1.88, 1.84, and 1.69 ppm and 1.92, 1.88, 1.82 and 1.69 ppm in a ratio ca. 1.8 : 1, Fig. 1C, assignable to two diastereomers; one diastereomer may be preferentially stabilized by H-bonding. The formation of Ir–S(O)G was confirmed by ESI-MS analysis giving a singly-charged ion peak centred at m/z 943.3, assignable to {(η⁵-Cp^xbiph)Ir(phpy)(S(O)G) + 2H}⁺ (calcd m/z 943.2, Fig. 2).

Fig. 1 ¹H NMR spectra for the reaction of NADH with Ir–SG, showing formation of NAD⁺ and sulfenato complex [(η⁵-Cp^xbiph)Ir(phpy)(S(O)G)]⁻ (Ir–S(O)G). (A) Formation of Ir–SG from reaction of Ir–py (0.6 mM) with GSH (3.5 mol equiv.) in 30% MeOD-d₄–70% PBS H₂O buffer; (B) 20 min after addition of NADH (2 mol equiv.) to the above equilibrium solution; (C) Formation of NAD⁺ and Ir–S(O)G, 2 h after addition of NADH. Peak assignments are shown.

Fig. 2 ESI-MS for an equilibrium solution containing Ir–SG and NADH in 30% MeOD-d₄–70% PBS H₂O buffer (v/v) 298 K. The peak at m/z 943.3 is assignable to the glutathione sulfenato complex {(η⁵-Cp^xbiph)Ir(phpy)(S(O)G) + 2H}⁺ (calcd m/z 943.2).

Table 1 Summary of the reaction of NADH with Ir–SG studied under three different conditions

Reaction	Buffer	Atmosphere	Products
1	Yes	Air	Ir–S(O)G, NAD^+
2	No	Air	Ir–S(O)G, NAD^+
3	Yes	N2	Ir–SG, NAD^+

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We showed previously that complex Ir–py can catalytically oxidize NADH to NAD⁺ by hydride transfer from NADH to Ir^III generating hydrido complexes (Ir–H) which can transfer the hydride to oxygen to form H₂O₂.¹² The production of H₂O₂ was confirmed by the appearance of a light blue colour on an H₂O₂ detection stick (Fig. 3), showing that ca. 60 μM of H₂O₂ was present after 48 h. The role of H₂O₂ in the oxidation of Ir–SG was further confirmed by addition of catalase which decomposes H₂O₂ to water and oxygen. As expected, no sulfenato complex Ir–S(O)G was observed after 4 h of reaction of Ir–SG with NADH in the presence of catalase, confirming that H₂O₂ is responsible for oxidizing Ir–SG to Ir–S(O)G.

Fig. 3 Detection of hydrogen peroxide. (A) To a solution of Ir–SG (produced by reaction of 0.6 mM Ir–py with 3.5 mol equiv. GSH in 30% MeOD-d₄–70% PBS H₂O buffer (v/v) at 310 K), 2 mol equiv. NADH were added. (B) After 48 h, H₂O₂ (about 60 μM) was detected by quantofix peroxide test sticks.

In the present case, the active hydrido species is probably formed from the small amounts of Ir–py (ca. 5% of Ir) present in solution in equilibrium with the more stable complex Ir–SG. The reaction appears to be catalytic, which is consistent with our previous results.¹² The sulfenato complex Ir–S(O)G has not been previously reported, and the only other known Ir sulfenato complex appears to be [IrCl₂(CH₃SO)(CO)L₂] (L = P(C₆H₅)₃ or P(C₆H₅)₂CH₃) prepared by the reaction of CH₃S(O)Cl with IrCl(CO)L₂.¹³

The reaction of NADH with Ir–SG was also studied in unbuffered aqueous solution, in air (Reaction 2), and under a nitrogen atmosphere in buffered solution (Reaction 3), Table 1. Importantly, no Ir–S(O)G was observed in the absence of NADH. However, addition of NADH to solutions under the conditions of Reaction 2 led to the generation of Ir–S(O)G, as for Reaction 1. In contrast, no Ir–S(O)G was formed after addition of NADH in Reaction 3 which was carried out under N₂ (Fig. S2†). These results indicate that: (1) buffer has little effect on the species formed, (2) Ir–S(O)G is formed only in the presence of O₂, indicating that the oxygen atom in Ir–S(O)G originates from O₂via the formation of H₂O₂, and (3) NADH plays a crucial role in the reaction, involving hydride transfer from NADH to Ir and finally to O₂.¹²

To confirm that H₂O₂ can directly oxidize Ir–SG to Ir–S(O)G, the oxidation of Ir–SG (produced by reaction of Ir–py with GSH) by H₂O₂ (10 mol equiv.) was studied at 310 K (Fig. 4). Ir–SG was oxidized to the sulfenato complex [(η⁵-Cp^xbiph)Ir(phpy)(S(O)G)]⁻ (Ir–S(O)G, Fig. 4B), and Ir–S(O)G was further oxidized to the sulfinato complex [(η⁵-Cp^xbiph)Ir(phpy)(S(O)₂G)]⁻ (Ir–S(O)₂G) after 24 h (Fig. 4C). These results were confirmed by ESI-MS analysis showing a singly-charged ion peak at m/z 943.2 (Fig. 4B), assignable to the glutathione sulfenato complex {(η⁵-Cp^xbiph)Ir(phpy)(S(O)G) + 2H}⁺ (calcd m/z 943.2), and a singly charged ion peak centered at m/z 981.1 (Fig. 4C), assignable to the sulfinato complex {(η⁵-Cp^xbiph)Ir(phpy)(S(O)₂G) + H + Na}⁺ (calcd m/z 981.2).

Fig. 4 ¹H NMR (methyl region, left) and ESI-MS spectra (right) showing the oxidation of Ir–SG by H₂O₂ in 30% MeOD-d₄–70% PBS D₂O buffer (v/v, pH* = 7.4) at 310 K, with formation of the sulfenato complex [(η⁵-Cp^xbiph)Ir(phpy)(S(O)G)]⁻ (Ir–S(O)G) and sulfinato complex [(η⁵-Cp^xbiph)Ir(phpy)(S(O)₂G)]⁻ (Ir–S(O)₂G). (A) Formation of Ir–SG produced by reaction of Ir–py (1 mM) with GSH (3.5 mM); (B) 15 h after addition of H₂O₂ (10 mol equiv.) to the above equilibrium solution, Ir–S(O)G and Ir–S(O)₂G were detected; (C) after 24 h, all Ir–S(O)G was further oxidized to Ir–S(O)₂G. The sample for ESI-MS was prepared in 30% MeOH–70% PBS H₂O buffer.

In summary, we have shown that the iridium glutathione adduct Ir–SG formed by substitution of the chlorido or pyridine ligands in half-sandwich C,N-chelated Ir^III biphenyltetramethylcyclopentadienyl by S-bound glutathione may be not a dead-end product if formed in cells from these anticancer agents. Complex Ir–SG can be readily oxidized to the sulfenato complex Ir–S(O)G and further to the sulfinate complex 1-S(O)₂G by H₂O₂ produced via hydride transfer from coenzyme NADH to iridium and then to O₂, Fig. 5.

Fig. 5 Possible reaction pathway for the oxidation of Ir–SG. *1-S(O₂)G is formed when an excess of H₂O₂ is added.

In future studies it will be interesting to investigate the possible role of these sulfenate and sulfinate complexes in the biological activity of iridium cyclopentadienyl complexes.

Acknowledgements

We thank the ERC (grant no. 247450), Science City (AWM/ERDF), and EPSRC for support, and members of EC COST Action CM1105 for stimulating discussions.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4qi00098f

‡ Current address: Department of Chemistry, University of Basel, Spitalstrasse 51, 4056 Basel, Switzerland.

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