Unexpected arene ligand exchange results in the oxidation of an organoruthenium anticancer agent : the first X-ray structure of a protein – Ru ( carbene ) adduct

The first X-ray structures of adducts formed between a RuII(N-heterocyclic carbene)(η6-p-cymene) compound and a protein are reported. Coordination to the protein induced the cleavage of the cymene ligand and EPR spectroscopy demonstrated the oxidation of the Ru centre.

To further support the positioning of the organometallic moieties, maps were generated using phenix.polder, 1 which allow for weak ligand density to be viewed in sharper detail (Fig. S4) and simulated annealing composite omit maps, which further proved the ligand identity (Fig. S5).

Synthesis
Compound 1 was prepared using the synthetic method described by Ott et al. 3 Protein crystallography HEWL crystals of 0.1-0.2mm were grown from a reservoir solution consisting of NaCl (0.8 M) and NaOAc (0.1 M, pH 4.7) which in the crystallisation drop was mixed with an equal volume of HEWL (100 mg/ml). 4Crystals formed within 24 h, and were then transferred into a drop of reservoir solution of NaNO 3 (0.8 M) and NaOAc (0.1 M, pH 4.7) supplemented with 1 (1.04 mg, 1 mg/mL).The crystals were observed to turn orange after 3 d, and X-ray analysis was performed after a period of 3 d and 1 month.For this purpose, the soaked crystals were transferred into a cryoprotectant of 20% glycerol, NaNO 3 (0.8 M) and NaOAc (0.1 M, pH 4.7), and flash frozen in liquid nitrogen.

Crystal data collection and analysis
X-ray diffraction data were collected on the crystallography beamlines MX1 at the Australian Synchrotron. 5Data were processed with XDS. 6 The protein crystallised in the space group P4 3 2 1 2. The structures were determined by molecular replacement using a monomer of lysozyme (PDB ID 4NHI) as a search model in PHASER. 7The models were refined with iterative rounds of refinement and model building in REFMAC 8 and COOT. 9Sodium ion placement was aided by the use of the WASP server at USF. 10 The anomalous difference maps were generated using FFT. 11The fragment formed was reformatted using Gaussian 12 (from the small molecule crystal structure of 1), while the final PDB and CIF files were generated by phenix.elbow. 13ectron paramagnetic resonance HEWL crystallisation was performed in EPR tubes (Wilmad 730-SQ-250M, 5 mm outer diameter) by mixing HEWL (1.25 ml, 100 mg/mL) and a crystallisation solution (0.7 M NaCl and 1.25 ml of 0.1 M NaOAc pH 4.7).The batch crystallization was performed over a period of 3 days, with crystals settling in the base of the EPR tube.The solution above the crystals was removed and the protein concentration was determined as 6.51 mg/mL using the molar absorptivity of HEWL and absorbance at 280 nm.The crystals were washed with the soaking buffer (0. EPR spectra were recorded using X band frequencies in a TE011 cavity of a JEOL JES-FA-200 spectrometer equipped with a variable temperature controller.Samples were cooled to 123 K and spectra were recorded at 9095.8 MHz with 100 kHz modulation, and 20 mW.Spectra of the buffer, and of crystals of HEWL and 1, as well as the control, KP1019, were recorded under the same conditions.Five spectra of the conjugate HEWL-1 were recorded and then co-added to improve the signal-to-noise ratio.Calculation of the integrated area for this spectrum was then divided by 5 for comparison of number of spins with the HEWL-KP1019 conjugate.The number of spins was calculated using the area obtained by doubly integrating the 1 st -derivative EPR spectrum eq.S1 × ( 4 ) × 6.02 × 10 23 where n = number of moles of CuSO 4 standard measured.The CuSO 4 spectrum was recorded at the same conditions except at one third of the gain; its integrated area was thus tripled to adjust for accurate calculation.
The spectra were simulated using the JEOL anisotropic simulation software.The HEWL-KP1019 spectrum is complex, showing more than one anisotropic signal.The g value range (axial and rhombic species g = 0.95-3.08)was consistent with those observed for KP1019 in other environments, as is the presence of more than one Ru III signal.

Mass spectrometry
Binding studies between 1 and His or HEWL were conducted with ESI-MS.His (1 mM) and 1 Table S2.Crystal data, collection, phasing and refinement.

Figure S1 .
Figure S1.Ru binding sites I and II as results from the reaction between HEWL and 1 overlaid with the native structure of HEWL (PDB ID 4NHI) shown in black.a) After 3 d (PDB ID 6BO1) and b) 1 month (PDB ID 6BO2) incubation periods, the positions of the residues Arg14 and His15 change as compared to the HEWL structure.c) Structural alteration of the Lys33 residue after 1 month of soaking and featuring the [Ru(1,3-dimethylbenzimidazol-2-ylidene)(OH x ) 2 Cl 2 ] adduct.The adjacent asymmetric unit is shown as a light teal cartoon in the background.d) Positional change of Trp108 after 1 month of soaking.

Figure S2
Figure S2 Details of binding site I for 1 on HEWL after 3 d incubation.The [Ru(dmb)(OH x )Cl 2 ] moiety is indicated in ball and stick representation with the Ru in teal.The electron density maps are contoured at 1σ (magenta and blue maps) while the anomalous difference maps are contoured at 4σ (yellow maps).a) Unbiased electron density (magenta) and anomalous difference map showing the site of interaction.b) Placement of the [Ru(dmb)(OH x )Cl 2 ] fragment into the refined electron density map (blue) with anomalous difference map, showing the bidentate modality of the interaction.

Figure S3 .
Figure S3.Ru binding sites as results from the reaction between HEWL and 1 and overlaid with the HEWL-Ru(η 6 -p-cymene)Cl 2 structure (PDB ID 5V4G) shown in black, highlighting the positional change of Arg14 after a) 3 d and b) 1 month soaking periods.

Figure S4 .
Figure S4.Details of binding site III detected in the HEWL structure soaked for 1 month with 1 showing a Ru ion sitting in the solvent channel.The Ru was identified to interact with the carbonyl O atom of Ala107.The electron density maps are contoured at 1σ (magenta and blue maps) while the anomalous difference maps are contoured at 4σ (yellow maps).a) Unbiased electron density (magenta) and anomalous difference map (yellow) showing the site of interaction.b) Placement of a [RuCl(OH x ) 4 ] fragment into the refined electron density map (blue) with anomalous difference map, showing the monodentate coordination mode.

Figure S5 .
Figure S5.Illustration of the octahedral Ru(NHC) adducts interacting with HEWL a) at site I through bidentate coordination involving Arg14 and His15, and b) at site II through monodentate coordination at Lys33.

Figure S6 .
Figure S6.Ru binding sites I and II with a map generated by phenix.polderwhich allows for ligand densities to be displayed with the exclusion of bulk solvent.a) For the crystal data resulting from a 3 d soak, a polder map was generated which was contoured to 3σ about the metal fragment [Ru(1,3-dimethylbenzimidazol-2-ylidene)(OH x )Cl 2 ] with Arg14 and His15 shown as a reference.b) For the structural data collected after 1 month of soaking a HEWL crystal with 1, a polder map was generated which was contoured to 2σ about the [Ru(1,3dimethylbenzimidazol-2-ylidene)(OH x )Cl 2 ] moiety with Arg14 and His15 shown as a reference.c) Polder map for crystal data for a 1 month soak contoured to 2σ about the [Ru(1,3dimethylbenzimidazol-2-ylidene)(OH x ) 2 Cl 2 ] ligand with Lys33 shown as a reference, while the adjacent asymmetric unit with Trp62 is given in light teal.d) Polder map contoured to 2σ about the [RuCl(OH x ) 4 ] moiety with Trp108 shown as a reference after soaking HEWL with 1 for 1 month.

Figure S7 .
Figure S7.Ru binding sites with maps generated by simulated annealing composite omit which validates the density surrounding the ligand.a) For the crystal data resulting from a 3 d soak, a simulated annealing composite omit map was generated which was contoured to 3σ about the metal fragment [Ru(1,3-dimethylbenzimidazol-2-ylidene)(OH x )Cl 2 ] with Arg14 and His15 shown as a reference.b) For the structural data collected after 1 month of soaking a HEWL crystal with 1, a simulated annealing composite omit map was generated which was contoured to 2σ about the [Ru(1,3-dimethylbenzimidazol-2-ylidene)(OH x )Cl 2 ] moiety with Arg14 and His15 shown as a reference.c) Simulated annealing composite omit map for crystal data for a 1 month soak contoured to 2σ about the [Ru(1,3-dimethylbenzimidazol-2-ylidene)(OH x ) 2 Cl 2 ] ligand with Lys33 shown as a reference, while the adjacent asymmetric unit with Trp62 is given in light teal.d) Simulated annealing composite omit map contoured to 2σ about the [RuCl(OH x ) 4 ] moiety with Trp108 shown as a reference after soaking HEWL with 1 for 1 month.

Figure S8 .
Figure S8.Mass spectrometric investigation of the binding of 1 to His.Zoom into the recorded spectrum for the His adduct [Ru(cym)(dmb)(His) -H] + and the calculated isotope pattern for the same peak.

( 1 : 1 )
were incubated at 25 °C for 6 d in acetate buffer (0.1 M, pH 4.7) or in 18.2 MΩ water.The samples were analyzed immediately after the start of incubation and after 3, 6, 24 h, 2, 3, and 6 d.For the protein interaction experiments HEWL (140 µM) and 1 (1 : 10) were incubated for 4 weeks and the samples were analyzed immediately after the start of incubation and after 1, 7, and 28 d.Both studies were conducted with a Bruker micrOTOF-Q II mass spectrometer in positive ion mode after 1 : 10 dilution of the samples with 30% acetonitrile/water containing 1% formic acid just before analysis.The Data Analysis 4.0 software package from Bruker Daltonics (Bremen, Germany) was used for processing of the data sets.

Table S3 .
Literature analysis of binding sites identified for Ru compounds on HEWL.