Stoichiometry effect on the structure, coordination and anticancer activity of gold(I/III) bisphosphine complexes

Abstract

Rationalizing the impact of oxidation states of Au-based complexes on function require synthetic strategies that allow for conserved molecular formula in Au(I) and their Au(III) counterparts. Oftentimes achieving Au(I) and Au(III) coordination complexes with the same ligand system is challenging due to the reactivity and stability of the starting Au(I) or Au(III) starting materials. Thus, attempts to study the impact of oxidation state on biological function has been elusive. We posit that Au complexes with the same ligand framework but different oxidation states will affect complex geometry and hence elicit differences in biological function or mechanism. In this work, we reacted 1,2-bis(diphenylphosphino)benzene with respective Au starting materials in different mole ratios to facilitate the synthesis of structurally distinct Au(I) or Au(III) complexes. Briefly, by reacting two stoichiometric equivalents of HAuCl₄·3H₂O or AuCl₃(tht) with one equivalent of 1,2-bis(diphenylphosphino)benzene, we obtained dicationic bis-[1,2-bis-(diphenylphosphino)benzene]gold(III) chloride whereas an equimolar ratio of HAuCl₄·3H₂O and 1,2-bis(diphenylphosphino)benzene gave the monocationic bis-[1,2-bis-(diphenylphosphino)benzene]gold(I) complex in moderate yield. The complexes were characterized spectroscopically by HRMS, RP-HPLC-MS, NMR and the purity ascertained by elemental analysis. The ³¹P NMR showed characteristic singlet peak at ∼22 ppm for the Au(I) complexes and ∼57 ppm for the Au(III) complexes. The structure of the Au(III) complexes was further confirmed by X-ray crystallography as a 5-coordinate Au(III) complex. Although both Au(I) and Au(III) complexes showed promising anticancer activity in MDA-MB-231 (breast cancer) and BT-333 (glioblastoma) cancer cell lines and inhibited maximal mitochondria respiration in MDA-MB-231 cells, the Au(III) complexes further induce ROS accumulation and facilitate depolarization of the mitochondria membrane potential in MDA-MB-231 cells. Taken together, the synthetic approach provides a way to elucidate the effect of Au(I)/Au(III) oxidation states on structure, activity, and potential mechanism with respect to the same ligand.

Fig. 1. Synthetic scheme showing the synthesis of Au(i/iii) DPPBz complexes. (a) Synthesis of Au(i) complexes from equimolar ratio of (HAuCl₄·3H₂O) and DPPBz with different counterions. (b and c) Synthesis of Au(iii) complexes from either tetrachloroauric(iii) acid trihydrate (HAuCl₄·3H₂O) or trichloro(tetrahydrothiophene)gold(iii) [AuCl₃(tht)].

Fig. 2. Crystal structure of the cation of 3-PF₆. Thermal ellipsoids are shown at the 50% probability level. Hydrogen and solvent molecules are omitted for clarity. Only one representative molecule from the asymmetric unit is shown.

Fig. 3. Solution stability of (a) 1-Cl and (b) 3-AuCl₄ in DMEM for 48 h. Reading was observed at λ = 260 nm.

Fig. 4. Gold bioaccumulation studies. (a) Whole cellular uptake of Au(i/iii) DPPBz complexes in MDA-MB-231 cancer cells. Cells were treated at a concentration of 10 μM for 18 h and Au concentration was determined by GFAAS. Data plotted as mean ± s.e.m (n = 3) (b) Mitochondria uptake of 1-Cl and 3-AuCl₄ in MDA-MB-231 cancer cells. Cells were treated at a concentration of 10 μM for 18 h and Au concentration was determined by GFAAS. Data plotted as mean ± s.e.m (n = 2). Ordinary one-way ANOVA, *P < 0.05 **P < 0.01, ***P < 0.001, ****P < 0.0001. ns = not significant.

Fig. 5. ROS determination. (a) Histogram and (b) Bar chart showing MDA-MB-231 cells treated with 1-Cl or 3-AuCl₄ at 5 and 10 μM for 2 h followed by staining with DCF-DA dye and the fluorescence intensity determined by flow cytometry at 488 nm. Ordinary one-way ANOVA *P < 0.05 **P < 0.01, ***P < 0.001, ****P < 0.0001. ns = not significant.

Fig. 6. Mitochondria bioenergetics study. (a) Seahorse assay of 1-Cl administered via pneumatic injection in vitro to MDA-MB-231 plated cells followed by addition of various inhibitors of ETC at various time point. (b and c) Key parameters extrapolated from Seahorse assay of 1-Cl. (d) Seahorse assay of 3-AuCl₄ administered via pneumatic injection in vitro to MDA-MB-231 plated cells followed by addition of various inhibitors of ETC at various time point. (e and f) Key parameters extrapolated from Seahorse assay of 3-AuCl₄. (g and h) Effect of oxidation state on maximal respiration and ATP production. Ordinary one-way ANOVA,*P < 0.05 **P < 0.01, ***P < 0.001, ****P < 0.0001. ns = not significant.

Fig. 7. MMP determination using TMRE dye. (a) Histogram and (b) Bar chart showing MDA-MB-231 cells treated with 1-Cl or 3-AuCl₄ at 5 and 10 μM for 90 minutes and fluorescence observed by flow cytometry. Ordinary one-way ANOVA,*P < 0.05 **P < 0.01, ***P < 0.001, ****P < 0.0001. ns = not significant.