Speciation of Transition-Metal-Substituted Keggin-Type Silicotungstates Affected by the Co-crystallization Conditions with Proteinase K

We report on the synthesis of the tetrasubstituted sandwich-type Keggin silicotungstates as the pure Na salts Na14[(A-α-SiW10O37)2{Co4(OH)2(H2O)2}]·37H2O (Na{SiW10Co2}2) and Na14[(A-α-SiW10O37)2{Ni4(OH)2(H2O)2}]·77.5H2O (Na{SiW10Ni2}2), which were prepared by applying a new synthesis protocol and characterized thoroughly in the solid state by single-crystal and powder X-ray diffraction, IR spectroscopy, thermogravimetric analysis, and elemental analysis. Proteinase K was applied as a model protein and the polyoxotungstate (POT)–protein interactions of Na{SiW10Co2}2 and Na{SiW10Ni2}2 were studied side by side with the literature-known K5Na3[A-α-SiW9O34(OH)3{Co4(OAc)3}]·28.5H2O ({SiW9Co4}) featuring the same number of transition metals. Testing the solution behavior of applied POTs under the crystallization conditions (sodium acetate buffer, pH 5.5) by time-dependent UV/vis spectroscopy and electrospray ionization mass spectrometry speciation studies revealed an initial dissociation of the sandwich POTs to the disubstituted Keggin anions HxNa5–x[SiW10Co2O38]3– and HxNa5–x[SiW10Ni2O38]3– ({SiW10M2}, M = CoII and NiII) followed by partial rearrangement to the monosubstituted compounds (α-{SiW11Co} and α-{SiW11Ni}) after 1 week of aging. The protein crystal structure analysis revealed monosubstituted α-Keggin POTs in two conserved binding positions for all three investigated compounds, with one of these positions featuring a covalent attachment of the POT anion to an aspartate carboxylate. Despite the presence of both mono- and disubstituted anions in a crystallization mixture, proteinase K selectively binds to monosubstituted anions because of their preferred charge density for POT–protein interaction.


General Information
All reagents and chemicals were of high-purity grade and were used as purchased without further purification. Na10[A-α-SiW9O34] was prepared according to the literature procedure. 1 Elemental analysis was carried out in aqueous solutions with 2 % HNO3 employing inductivecoupled plasma mass spectrometry (PerkinElmer Elan 6000 ICP-MS) and atomic absorption spectroscopy (PerkinElmer 1100 Flame AAS). Standards (Merck, Ultra Scientific and Analytika Prague) were prepared from 1000 mg/L single-element standard solutions.
Attenuated total reflection Fourier−transform Infrared Spectroscopy (ATR FTIR): All spectra were recorded on a Bruker Vertex70 IR Spectrometer equipped with a single−reflection diamond−ATR unit. Frequencies are given in cm -1 , intensities denoted as w = weak, m = medium, s = strong.
Thermogravimetric analysis (TGA): TGA was performed on a Mettler SDTA851e Thermogravimetric Analyzer under N2 flow with a heating rate of 5 K min -1 in the region 298−973 K.
Single crystal X-ray diffraction (SXRD): The X-ray data were measured on a Nonius Kappa-CCD diffractometer, equipped with a 0.3 mm monocapillary optics collimator, graphite monochromatized MoKα-radiation, at 200 K (Na{SiW10Co2}2 and Na{SiW10Ni2}2) and on a Bruker D8 VENTURE equipped with a multilayer monochromator, Mo Kα Incoatec Microfocus sealed tube, and Kryoflex cooling device at 140 K ({SiW9Co4}). The structures were solved by direct methods and refined by full-matrix least-squares. Non-hydrogen atoms were refined with anisotropic displacement parameters. The following software was used for the structuresolving procedure: frame integration, Bruker SAINT software package using a narrow-frame algorithm (absorption correction) 2 , SADABS 3 , SHELXS-2013 4 (structure solution), SHELXL-2013 5 (refinement), OLEX2 6 (structure solution, refinement, molecular diagrams, and graphical user-interface), and SHELXLE 7 (molecular diagrams and graphical user interface). Experimental data are provided in Table S4.
Powder X-ray diffraction was performed on an EMPYREAN diffractometer system using CuKα radiation (λ = 1.540598), a PIXcel3D-Medipix3 1 × 1 detector (used as a scanning line detector) and a divergence slit fixed at 0.1 mm. The scan range was from 5° to 50° (2θ).
UV/Vis spectroscopy: UV/Vis spectra were collected on a Shimadzu UV 1800 spectrophotometer equipped with a Julaba F25 water bath to ensure a constant temperature of 20 ± 2°C in the sample cuvette, mimicking the temperature of the crystallization experiments.
Electrospray ionization mass spectrometry (ESI MS): ESI MS was performed with an ESI−Qq−oaRTOF supplied by Bruker Daltonics Ltd. Bruker Daltonics Data Analysis software was used for peaks assignment. The measurement was carried out in H2O and NaOAc / HOAC buffer diluted with 50 % of ACN, collected in negative and positive ion mode and with the spectrometer calibrated with the standard tune−mix to give an accuracy of ca. 5 ppm in the region of m/z 300−1900. The signals with low intensities show an accuracy of ca. 10 ppm. Figure S1. Key milestones in the application of POTs in protein crystallography. For the timeline, investigation, which, in the author's opinion, gave impetus to the further development of the POT's application in protein crystallography, are highlighted [8,9,10,11,12,13,14,15,16,17,18,19]. The names of the first authors are shown in black, the solid POT in purple, the POT anion detected in crystal structure in blue, the protein and PDB entries in green.

Synthesis of polyoxotungstates
To a vigorously stirred solution of Co(OAc)2 • 4 H2O (1.5 g, 6 mmol) in water (75 ml) Na10[Aα-SiW9O34] (5 g, 2 mmol) was added in small portions followed by stepwise heating of the reaction mixture to 80°C. After addition of 50 mL of water and reaction at 80 °C for 1 h, the reaction mixture was cooled to room temperature and passed through a cation-exchange column (AMBERLITE™ IR120 resin, Lenntech) which was equilibrated with water. Addition of a solution of NaCl (1.6 g) in 6.25 mL water to the collected eluate and slow evaporation of the filtered solution at room temperature led to formation of dark red rod-shaped crystals of Na{SiW10Co2}2 after four weeks. Yield: 2.5 g, 20% based on W. Anal. Calcd. (%) for Na 14  The acetato-capped tetra-Co(II) POT {SiW9Co4} was synthesized as described by Lisnard et al. in [20] and characterized by IR-spectroscopy ( Figure S2) and SXRD (Table S4). Figure S2. IR analysis of Keggin POT derivatives. The α-Keggin Sandwich derivatives Na{SiW10Co2}2 and Na{SiW10Ni2}2 feature slight impurities of acetate salt with signals at 1558 and 1412 cm -1 (correspond to orange spectra of Na{SiW10Co2}2). These signals are strongly pronounced for the acetate-bridged POT {SiW9Co4}. As can be expected from the shared W-O core structure, the IR spectra are highly similar for all Keggin POT derivatives. 21 For assignment of characteristic vibrations of the Keggin POT scaffold, see Table S1. ν: vibration, δ: distortion, M: transition metal.      The architecture of the isostructural Na{SiW10Ni2}2 and Na{SiW10Co2}2 represents a dimer of [α-SiW10O37] 10ions (Figure S5 A, B). The overall atom connectivity in Na{SiW10M2}2 features a central square-shaped motif of two M II centers and two µ3-OH. Thereby, all four M II atoms are linked through only two µ3-oxygens. Since the half-units are related by an inversion center, there are only two structurally non-equivalent types of M II centers exhibiting a distorted octahedral coordination environment with M-O distances ranging from 2.001(6) to 2.27(3) Å, which is in accordance with the findings reported by Haider et al. 22 In Na{SiW10M2}2, each α-SiW10O37 unit bears one labile water ligand and a very reactive µ3-OH site, as well as six basic µ2-O positions bridging W VI and Co II or Ni II . These oxo-sites show a pronounced protonation propensity as indicated by bond valence sum calculations (BVS) ( Table S5). Figure S5. Structures of Keggin POT derivatives Na{SiW10Co2}2 (A), Na{SiW10Ni2}2 (B), {SiW9Co4} (C) in the solid state applied for co-crystallization with proteinase K. Color code: dark blue, W; red, O; rose, Co; green, Ni; ivory, Si; grey, C. All POT structures feature labile aquo or acetato ligands (marked by circles) as well as highly nucleophilic and basic bridging oxo-sites. Figure S6. Comparison of the experimental and simulated PXRD patterns of Na14[(A-α-SiW10O37)2{Co4(OH)2(H2O)2}] • 37 H2O Na{SiW10Co2}2. Given the loss of crystal water already occurring at room temperature (please see Figure S3, along with Table S2), the presence of more than one crystalline phase belonging to the investigated POTs can be suggested, which gave rise to the observed difference in the experimental and simulated patterns. Figure S7. Comparison of the experimental and simulated PXRD patterns of Na14[(A-α-SiW10O37)2{Ni4(OH)2(H2O)2}] • 77.5 H2O Na{SiW10Ni2}2. Given the loss of crystal water already occurring at room temperature (please see Figure S4, along with Table S3), the presence of more than one crystalline phase belonging to the investigated POTs can be suggested, which gave rise to the observed difference in the experimental and simulated patterns.      Scheme S1. Rearrangement pathway for dimeric sandwich POTs based on time-dependent ESI MS (Figures S13, S14) and UV/vis studies (Figures S10, S11). Starting from the tetrasubstituted dimeric sandwich POT Na{SiW10M2}2, disubstituted monomeric species {SiW10M2} after 1) 1 day in H2O and 2) 1 day in 100 mM NaOAc/AcOH (pH 5.5). The disubstituted monomeric representatives further hydrolyze to monosubstituted monomers α-{SiW11M} after incubation 3) in 100 mM NaOAc/AcOH (pH 5.5) for one week, whereas no change is observed in the pristine disubstituted monomers {SiW10M2} after 4) one week in H2O (M = Co II , Ni II ). Black, blue and red spheres represent the Si IV , M II and oxygen ions, respectively. Magenta octahedra for {WO6}. The exact pathway for {SiW9Co4} cannot be identified by ESI-MS due to the presence of the acetate group in the POT structure; however, the time-dependent UV/Vis spectra ( Figure S10B) in sodium acetate buffer clearly indicate POT rearrangement.

Preparation of Proteinase K for crystallization
Standard chemicals at least of analytical grade (Sigma Aldrich) were used throughout this study. Proteinase K from Tritirachium album was purchased from Sigma-Aldrich (P6556), dissolved in 10 mM Tris/HCl pH 7.0, 0.05 % (m/v) NaN3 to obtain a 100 g/L protein solution and centrifuged for 15 min at 20817 x g (14000 rpm) to spin down traces of insoluble material. The supernatant was considered sufficiently pure for crystallization trials without further purification (Figures S15, S16).  After few hours, a fine protein precipitate formed due to unspecific aggregation, from which spindle-shaped crystals appeared after 2 weeks. These crystals effected resolubilization of precipitated protein, which then fed crystal growth leading to 50-100 µm crystals. An alternative crystallization condition consisted of 100 mM NaOAc/AcOH pH 5.5 with 0.3-0.7 M (NH4)2SO4 and 0.5 M betaine. The zwitterionic additive betaine was successfully applied to prevent the strong unspecific charge-driven aggregation observed for concentrated POT-protein mixtures in the acidic milieu and facilitate the growth of large, ordered crystals, which helped in co-crystallization with Na{SiW10Ni2}2. Protein crystals were harvested in nylon loops, quickly wiped through a cryo-protectant solution containing 15% (v/v) glycerol and flash-frozen in liquid nitrogen. The crystals were analyzed with a Bruker D8 VENTURE X-ray diffractometer equipped with a multilayer monochromator, a PHOTON II charge-integrating pixel array detector, a CuKα Incoatec Microfocus (sealed tube) and a Kryoflex cooling device. For data collection and refinement statistics refer to   [d] Percent correlation between random half-sets of anomalous intensity differences.
[f] Rfree is calculated using a randomly chosen reference set of 5% of all the reflections collected for each data set.

Binding positions of Keggin anions
The two main interaction positions of Keggin-POTs on the surface of proteinase K (Figue S17) are involved in various interaction modes with non-polar, positively and negatively polarized surface patches. The two Keggin POTs substituted by Co II and Ni II (monosubstituted α-{SiW11Co} and α-{SiW11Ni}) shared a common position on the proteinase K surface, forming a covalent bond to the aspartate side-chain D207 (position 1) (Figure S18A). The bridging contacts to three different protein molecules are depicted in Fig.  S10A. The Keggin anions were also observed in the vicinity of the residue S45 (position 2) in a second common site. In this position, the POT clusters interacted with two distinct protein molecules (Figure S18B).       (Figure 2) for the protein crystal structures with α-{SiW11Co} (PDB entries: 7A9F and 7A9M) and α-{SiW11Ni} (PDB entry: 7A9K), including hydrogen bonds. The POT atoms are numbered according to Figure S19.  Table S9. Stabilizing protein interactions within the hydrogen-bonding network at position 2 (Figure 3) for the protein crystal structures with α-{SiW11Co} (PDB entries: 7A9F and 7A9M) and α-{SiW11Ni} (PDB entry: 7A9K). The POT atoms are numbered according to Figure S19.