New activation mechanism for half-sandwich organometallic anticancer complexes

Half-sandwich RhIII anticancer complexes with activated Cp* rings not only undergo sequential CH3 H–D exchange, but also react with biological dienes, generating RhI Diels–Alder adducts in aqueous media at ambient temperature.


S.3. Electrospray ionization mass spectrometry (ESI-MS)
Positive ion electrospray mass spectra were obtained on a Bruker Daltonics Esquire 2000 ion trap mass spectrometer. All samples were prepared in methanol (100%). Data were processed using Data-Analysis version 3.3 (Bruker Daltonics).
HR-MS analysis was carried with a Bruker MaXis plus Q-TOF mass spectrometer equipped with electrospray ionisation source. The mass spectrometer was operated in electrospray positive ion mode with a scan range 50-2,400 m/z. Source conditions were: end plate offset at -500 V; capillary at -4000 V; nebulizer gas (N2) at 0.5 bar; dry gas (N2) at 4 L/min; dry temperature at 453 K. Ion tranfer conditions as: ion funnel RF at 200 Vpp; multiple RF at 200 Vpp; quadruple low mass set at 50 m/z; collision energy at 5.0 eV for MS and 10-20 eV for MS/MS, MS/MS isolation window 10; collision RF at 500-2000 Vpp; transfer time set at 50-150 µs; pre-pulse storage time set at 5 µs. Calibration was carried out with sodium formate (10 mM) before analysis.

S.4. nESI-FT-ICR mass spectrometry
Samples were analyzed via nano-electrospray ionization (nESI)-Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS). All experiments were carried out on a Bruker SolariX FT-ICR Mass Spectrometer, fitted with a 12 tesla actively shielded magnet (Bruker Daltonik GmbH, Bremen, Germany). Samples were diluted 1000-fold from NMR samples/reaction mixtures with appropriate deuterated/non-deuterated solvents (to ~1µM), an aliquot of each sample (10-20 µL) was ionized from ~1 µm glass nESI capillaries using a capillary voltage of 900-1200V, ions were accumulated for 0.01 s in the hexapole-based collision cell before transfer to the ICR cell for detection. Ions of m/z 147-3000 were excited using a frequency sweep excitation and detected for 3.2 s transient length (8M (16-bit) data points), producing a resolving power of ~850,000 (at 400 m/z) for all spectra. Mass spectra were externally calibrated using a quadratic calibration function/internally using single point calibrations and then manually interpreted and assigned via Data Analysis v4.2 (Bruker Daltonik GmbH, Bremen, Germany).

S.5. X-ray crystal structures
Diffraction data were collected on an Oxford Diffraction Gemini four-circle system with a Ruby CCD area detector. All structures were refined by full-matrix least squares against F 2 using SHELXL 97 3 and were solved by direct methods using SHELXS 4 (TREF) with additional light atoms found by Fourier methods. Hydrogen atoms were added at calculated positions and refined using a riding model. Anisotropic displacement parameters were used for all non-H atoms; H-atoms were given an isotropic displacement parameter equal to 1.2 (or 1.5 for methyl) times the equivalent isotropic displacement parameter of the atom to which they are attached. The data were processed by the modelling program Mercury 1.4.1. The Rh-Cl distances are in the range 2.385-2.403 Å, with complexes containing extended Cp x rings showing slightly longer bond lengths. The Rh-Cp x centroid distance of 1.778-1.795 Å shows little change with extension of the Cp* ring. Interestingly, complexes 6, 7 and 9 have one Rh-N distance slightly longer than the other. For complex 12, a suitable crystal was selected and mounted on a glass fibre with Fromblin oil and placed on an Xcalibur Gemini diffractometer with a Ruby CCD area detector. The crystal was kept at 150(2) K during data collection.
Using Olex2, 5 the structure was solved with the ShelXT 6 structure solution program using Intrinsic Phasing and refined with the ShelXL 7 refinement package using Least Squares minimization. The asymmetric unit contains the Rh complex, two PF6 counter ions and a partially occupied water molecule. No hydrogens were located on the water molecule. The complex was modeled as disordered over two closely related positions. The occupancy was originally linked to a free variable that settled around 85:15 and the occupancy of the components was fixed at this value for the rest of the refinement. The PF6 counter ion (P20_F26) was modeled as disordered over two closely related positions again linked to 85:15 occupancy. Additionally, the major component was also modeled as disordered where the components were related by a rotation about the meridian. The occupancy of these components (F23-F26: F23A-F26A) was linked to a free variable which refined to 76:24 (of the 85% occupancy). Minor components were refined isotropically and several DFIX, DANG and SIMU restraints were used to give the minor components reasonable bond lengths, angles and thermal parameters.

S.6. pH* measurements.
pH* values (pH meter reading without correction for the effect of deuterium) were determined at ambient temperature using a minilab IQ125 pH meter equipped with a ISFET silicon chip pH sensor and referenced in KCl gel. The electrode was calibrated with Aldrich buffer solutions of pH 4, 7 and 10. pH* values were adjusted with NaOD or DNO3 solutions in D2O.

S.7. Elemental analysis.
Elemental analyses were performed by Warwick Analytical Service using an Exeter Analytical elemental analyzer (CE440).

S.8. DFT calculations
The electronic energies for all complexes under study were calculated with Gaussian 09, version D. 8 using CAM-B3LYP functional 9 and CEP-31G basis set. 10-12 For comparison, the Ir and Rh complexes were modeled with the TPSSh functional 13 and QZVP basis set 14,15 for both M(Cp* -)(bpy)OH and M([Me4Cp=CH2] 2-)(bpy)H2O isomers. In each case the structure was optimized using the standard optimization procedure of Gaussian 09, followed by the frequency calculations in order to find whether the true minimum was found. Ultrafine grid of integration was applied in all cases. In the case of M([Me4Cp=CH2] 2-)(bpy)H2O isomers calculated with TPSSh/QZVP method, the calculation of forces (Gaussian keyword CALCF) was applied. The proton transfer transition state and cyclopentadienyl anion rotation barrier for [Rh(Cp -)(bpy)OH] was calculated using the QST3 method, followed by frequency calculations.

Hydride mechanism
A mechanism for H/D exchange that involves formation of a hydride intermediate was also considered. Such a mechanism operates for Cp*Ir III catalyzed H/D exchange of aromatic CH bonds in benzene derivatives in solvents such as MeOD-d4. 16 H2 was found to remain coordinated to Ir and is rather loosely bound to Rh. In both cases the H2 isomer is greatly destabilized relative to the hydride complex (by 190 kJ mol -1 and by 120 kJ mol -1 for Ir and Rh, respectively). If it is assumed that the deuteration of Cp* in D-hydride complex proceeds via formation of coordinated HD, then this intermediate state presents a very high-activation barrier. Therefore this mechanism did not seem to be competitive with the Rh-OH mechanism.

Effect of solvent
Optimization calculations were also performed using methanol as solvent with the polarisable continuum model (with IEFPCM keyword of Gaussian). Frequency calculations confirmed that true minima were identified. The sequence of the energy differences for the hydroxide/aqua pairs Rh(bpy)Cp*OH/H2O, Ir(bpy)Cp*OH/H2O and Rh(en)Cp*OH/H2O pairs is qualitatively the same as in the absence of methanol. The energy differences are in the This is an interesting structure (pdb files Rh_bpy_OH_MeOH and Rh_bpy_H2O_MeOH), a possible active state which may revert to the initial complex, with methanol exchanging the proton. Interestingly, the second species lie 54 kJ/mol higher in energy than the first, while the previously quoted difference for solvent-modelled [Rh(bpy)(Cp*)OH/H2O] pair of 49 kJ/mol is very similar. These data show that the inclusion of solvent has little effect on the general conclusions discussed in the main text.

M(III) versus M(I) intermediates
Comparison of the geometries of a) free deprotonated Cp* ligand (optimized with CAM-
Integration of a corresponds to 13H. c) After incubation for 15 h after addition of NaCl.