Interactions of oxaliplatin with the cytoplasmic thiol containing ligand glutathione

Abstract

The complexation of the Pt-based anti-cancer drug oxaliplatin (OxPt) with biological ligands other than DNA is believed to be a major cellular sink for the drug reducing its therapeutic effect and acting as a potential cause of toxicity. In this paper, linear ion trap electrospray ionization mass spectrometry was employed to study the interaction of oxaliplatin with the cytoplasmic thiol containing tripeptide ligand γ-L-glutamyl-L-cysteinyl-glycine (GSH) this being the most abundant low-molecular-weight thiol containing molecule in human cells. Evidence of protonated dimers and multimers of oxaliplatin, protonated multimers of glutathione as well as several different combinations of these protonated species is presented. Most species observed were unambiguously assigned and compared to their theoretical isotopic patterns. Fragmentation of the collisionally-activated protonated complex of glutathione with oxaliplatin [GSH + OxPt + H]⁺ resulted in the formation of several species. No experimental evidence for [GSH + H]⁺ formation from the [OxPt + GSH + H]⁺ precursor was observed. Density functional calculations at B3LYP/LANL2DZ were used to obtain structural information and relative free energies of different isomers of the observed precursor [OxPt + GSH + H]⁺ both in the gas phase and in solution as well as to probe its fragmentation, highlighting mechanisms that account for all the experimental results. Data are presented to show several binding modes between electron rich sites such as S, N, O centers of GSH and the Pt metal of oxaliplatin. Calculations were also employed to obtain proton affinities and free energies of key reactions. The proton affinities of GSH and OxPt at 298 K were calculated to be 255.3 and 233.5 kcal mol⁻¹ respectively. The enthalpy and free energy, based on the most thermodynamically favored conformers of the reactants and products, for the addition reaction [Pt(dach)]²⁺ + [GSH − H]⁻ → [GSH − H + Pt(dach)]⁺ (where dach represents diaminocyclohexane) in the gas phase at 298 K were determined to be −311.3 and −290.2 kcal mol⁻¹ respectively. Similarly, the enthalpy of the gas phase reaction [Pt(dach)]²⁺ + GSH → [GSH + Pt(dach)]²⁺ at 298 K was determined to be −169.2 kcal mol⁻¹.