1  

Supporting information

Transition metal-substituted Keggin polyoxotungstates enabling covalent attachment to proteinase K upon co-crystallization

Joscha Breibeck [a], Aleksandar Bijelic [a] and Annette Rompel [a] *

[a] Dr. Joscha Breibeck, Dr. Aleksandar Bijelic, Annette Rompel, Universität Wien, Fakultät für Chemie, Institut für Biophysikalische Chemie, Althanstraße 14, 1090 Wien, Austria; http://www.bpc.univie.ac.at

*Correspondence to: annette.rompel@univie.ac.at

Electronic Supplementary Material (ESI) for Chemical Communications.This journal is © The Royal Society of Chemistry 2019

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Contents

1. Proteinase K treatment before crystallization .............................................................................. 3 

2. Keggin POT synthesis and characterization ................................................................................ 3 

2.1 Synthesis ..................................................................................................................................... 3 

2.2 Characterization ......................................................................................................................... 6 

3. Crystal structures of proteinase K with Keggin POTs ................................................................ 9 

3.1. Protein crystallization ............................................................................................................... 9 

3.2. X-ray data collection, structure solution and refinement ................................................... 10 

3.3. Main positions of Keggin anions ........................................................................................... 12 

3.4. Additional positions and conformations of Keggin anions ................................................ 17 

3.5. Keggin POT occupancies in the crystal structures ............................................................ 18 

4. Structural integrity of proteinase K upon prolonged storage with a Keggin POT ................. 19 

4.1. SDS-PAGE analysis of proteinase K incubated with a Keggin anion ............................. 19 

4.2. Enzymatic activity assay in the presence of a Keggin anion ........................................... 19 

5. CCDC data base survey on monosubstituted Keggin POTs with ligands at the transition metal center ......................................................................................................................................... 21 

6. Acknowledgements ........................................................................................................................ 25 

7. References ...................................................................................................................................... 25 

 

 

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4  

transition metal salt as described in [2]. (NH4)6[β3-SiW11O39{Co(H2O)}] · 18.5 H2O was obtained by treatment of [β3-SiW11O39]

8- [3] with Co2+ according to [2]. The K+ counter cations of the POT products were exchanged to NH4

+ using AMBERLITE™ IR120 resin (Lenntech), which dramatically increased their aqueous solubility and facilitated preparation of concentrated stock solutions up to 20 mM.

Table S1: Keggin POTs used in this study. The exact amount of hydration water of the Keggin compounds was determined by TGA (Table S3). OAc: acetate.

Keggin POT compound Compound

abbreviation

Synthesis according

to

Transition metal salt used for

synthesis (NH4)5[α-PW11O39{Co(H2O)}] · 26.5 H2O α-[PW11Co]5- [1], [2] Co(OAc)2 · 4 H2O

(NH4)5[α-PW11O39{Ni(H2O)}] · 19.5 H2O α-[PW11Ni]5- [1], [2] Ni(NO3)2 · 6 H2O

(NH4)5[α-PW11O39{Cu(H2O)}] · 16.5 H2O α-[PW11Cu]5- [1], [2] Cu(OAc)2 · 4 H2O

(NH4)5[α-PW11O39{Zn(H2O)}] · 19 H2O α-[PW11Zn]5- [1], [2] Zn(OAc)2 · 2 H2O

(NH4)6[β3-SiW11O39{Co(H2O)}] · 18.5 H2O β-[SiW11Co]6- [2], [3] Co(OAc)2 · 4 H2O

All Keggin POT anions used for co-crystallization with proteinase K are summarized in Fig. S2. The basic α-Keggin polyoxometalate archetype comprises twelve oxygen-sharing {WO6} units arranged in tetrahedral symmetry around a templating XO4 tetrahedron, where X can be a main group or transition element suitable in size. By successive rotation of the four {W3O13} triad building-blocks by 60 °, four more Keggin isomers can be derived (β, γ, δ, and ε), with decreasing energetic preference. Therefore, only the most stable α- and β-Keggin isomers are of practical relevance for applications in aqueous solutions as the other isomers require organic solvents or conjugation to organic ligands for stabilization[4]. Interestingly, the β-isomer stability increases with decreasing positive charge of the central heteroatom, resulting in a remarkable share of the [SiW12O40]

4- anion adopting the β-configuration[3]. Both the α- and the β-Keggin structure can be partially hydrolyzed to mono-lacunary clusters and another metal ion, again suitable in size, can be inserted into the octahedral cavity, which serves as a stable multidentate inorganic ligand.

While the removal of any single WVI center from the Td-symmetric α-isomer leads to only one lacunary α-form, the less symmetric β-isomer yields three different lacunary structures β1, β2 and β3, depending on the kind of WVI removed (Fig. S2). Substitution of single WVI ions by less positively charged transition metal ions usually leads to an overall increase of the Keggin POT anionic charge and hydrolytic stability, strongly suggesting application in solution studies including protein crystallography. The low-charge transition metals MII usually complete their octahedral coordination sphere by a labile terminal aquo-ligand rather than an oxo-ligand to avoid local excess of negative charge. This water ligand displays the most reactive site of a substituted Keggin anion as it can be replaced by any stronger inorganic or organic ligand.

The distinguishable groups of oxygen atoms in the Keggin framework (Fig. S2) feature various degrees of reactivity in terms of nucleophilicity and relative basicity. Both generally increase with the number of metal centers bound to the respective oxygen atom: Ot < µ2-O (< µ4-O, sterically shielded). The terminal oxygens are most strongly bound to the WVI centers and chemically most inert, but still capable of accepting H-bonds. On the other hand, the bridging µ2-oxygens are considered as potential protonation sites and therefore stronger H-bond acceptors, and even H-donors upon protonation. Precisely looking, there

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are two symmetrically different sorts of µ2-O atoms in the Keggin structure, and the bridging oxygens within the same metal triad are calculated to be slightly more basic than those connecting the triads[5]. Their reactivity is further enhanced by connection to more electron-rich transition metals M: W-µ2-O-W < W-µ2-O-M[6]. Such bridging oxygens can even serve as conjugation sites for organic ligands due to their pronounced nucleophilicity[7].Taken together, transition metal substitution is expected to increase the versatility of chemical interactions and to activate the rather inert Keggin POT surface.

Fig. S2: Structures of Keggin POTs used in this study as revealed from protein co-crystals. The connectivity of the POT architecture is visualized by an overlay of a ball-and-stick and a transparent polyhedron model. The central P (yellow) or Si (ivory) tetrahedron is enclosed by twelve tungsten atoms, shown as big blue spheres and surrounded by light blue octahedra, the corners of which are formed by oxygen atoms (red spheres) of various kinds (bridging or terminal). The substituted transition metals (M) are depicted in colors referring to their octahedral complexes (Co: red, Ni: green, Cu: cyan, Zn: grey), and this color-code is conserved in all following figures. As revealed by the protein-POT co-crystal structures presented here later, only the Co- and Ni-containing POTs featured an aquo-ligand in distorted octahedral geometry, whereas the Cu- and Zn adopted a distorted square-pyramidal geometry without such a ligand. This finding is further supported by the measured µ4-O-metal distances (encircled in white) being clearly longer in the latter geometry. The β-isomer differs from the α-form by rotation of the upper W-W-M triad by 60 °. Depending on the substitution site within

the β-Keggin structure, three different isomers (β1, β2 and β3) can be obtained[3], with the here depicted

β3-isomer being used in this study.

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2.2 Characterization

All substituted Keggin polyoxotungstates were confirmed by thorough compositional analyses (elemental analysis, Table S4; ESI MS, Table S5) and structural characterization (IR, Table S2; NMR, Table S6).

Infrared absorption spectra (Fig. S3, Table S2) were collected in the range 4000−400 cm−1 using a Bruker Vertex 70 IR Spectrometer equipped with a single-reflection diamond-ATR unit.

Fig. S3: IR analysis of Keggin POTs. The four P-centered α-Keggin isomers are characterized by a strong P-O vibration close to 1000 cm-1. As can be expected from the shared W-O core structure, the IR spectra are highly similar for all Keggin POTs. ν: vibration, δ: distortion, M: transition metal.

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To identify the water content of the solid Keggin POTs, thermogravimetric analysis (TGA, Table S3) was applied with a Mettler SDTA851e thermogravimetric analyzer under nitrogen flow with a temperature ramp of 5 K min−1 from 298−1023 K.

Elemental analysis was carried out in aqueous solutions with 2 % HNO3 employing inductive-coupled 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.

Electrospray ionization mass spectrometry (ESI-MS, Table S5) was conducted with an ESI-Qq-oaRTOF from Bruker Daltonics Ltd., and data deconvolution and analysis were performed using the Bruker Daltonics Data Analysis software. Keggin POTs were dissolved in a 1:1 mixture of water/MeCN, and mass spectra were collected in negative ion mode after calibration with the standard tune-mix to give an accuracy of ca. 5 ppm in the m/z region of 300−3000.

31P NMR analysis (Fig. S4, Table S6) of the Keggin POTs was performed with 20 mM POT compound in 100 mM NaOAc/AcOH pH 5.5 with 90% D2O. Spectra were recorded with a Bruker FT-NMR spectrometer Avance Neo 500 MHz at 242.97 MHz and 25 °C using standard procedures. Chemical shifts were measured relative to external 85% H3PO4.

Fig. S4: 31P NMR analysis of Keggin POTs. For POTs with the paramagnetic transition metals CoII, NiII and CuII, comparably broad signals were identified around +460 and -39 ppm. The signal broadening and reduced intensities arise from additional coupling effects involving unpaired d-electrons from the transition metals[8]. Only the diamagnetic ZnII ion yields an intense and sharp phosphorus signal. Importantly, signals for free phosphate ions and for the transition-metal-free α-[PW11O39]

7- are completely absent (cf. Table S6), confirming the purity of the Keggin POTs as well as their solution stability at the applied pH conditions (pH 5.5).

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Table S2: IR analysis of Keggin POTs. Due to their structural similarity, the POT anions yielded strikingly similar signals. The strong P-O vibration is characteristic for all phosphate-containing compounds. ν: vibration, δ: distortion, M: transition metal.

Keggin POT compound Wavenumber (cm-1) (NH4)5[α-PW11O39{Co(H2O)}] · 26.5 H2O 1075/1051 990 951 879 793 744 692/664 620 610 505/485 (NH4)5[α-PW11O39{Ni(H2O)}] · 19.5 H2O 1058 996 956 879 794 758 695 621 610 507/487 (NH4)5[α-PW11O39{Cu(H2O)}] · 16.5 H2O 1000 936 905 879 772 736 668 621 610 489 (NH4)5[α-PW11O39{Zn(H2O)}] · 19 H2O 1024 965 931 877 824 750 698 621 610 502/489/473 (NH4)6[β3-SiW11O39{Co(H2O)}] · 18.5 H2O - 996 950 879 807 737 681 620 610 536/511/483 Signal assignment ν P-O ν W=O ν,δ W-O-W/W-O-M ν,δ W-O-W ν,δ W-O-W/W-O-M Table S3: Thermogravimetric analysis of Keggin POTs. The amount of hydration water of the Keggin compounds was determined previous to elemental analysis. MM: molecular mass.

Keggin POT compound MM (POT) Mass % H2O Mass % POT MM (POT·n H2O) MM (n H2O) n (H2O) n (final) (NH4)5[α-PW11O39{Co(H2O)}] · 26.5 H2O 2736.12 15.31 84.69 3230.75 494.63 27.5 27.5 (NH4)5[α-PW11O39{Ni(H2O)}] · 19.5 H2O 2735.88 11.82 88.18 3102.61 366.73 20.4 20.5 (NH4)5[α-PW11O39{Cu(H2O)}] · 16.5 H2O 2740.73 10.43 89.57 3059.87 319.14 17.7 17.5 (NH4)5[α-PW11O39{Zn(H2O)}] · 19 H2O 2742.58 11.59 88.41 3102.12 359.54 20.0 20 (NH4)6[β3-SiW11O39{Co(H2O)}] · 18.5 H2O 2733.23 11.46 88.50 3087.00 353.80 19.7 19.5 Table S4: Elemental analysis of Keggin POTs. Elemental composition was verified at least by the expected tungsten mass percentage corresponding to 11 tungsten atoms. The experimentally found mass percentages (in %) are listed for each element, and the expected values are given in brackets for comparison.

Keggin POT compound W P Si Co Ni Cu Zn (NH4)5[α-PW11O39{Co(H2O)}] · 26.5 H2O 63.1 (60.9) 0.90 (0.93) - 1.44 (1.77) - - - (NH4)5[α-PW11O39{Ni(H2O)}] · 19.5 H2O 58.3 (63.3) 0.86 (0.97) - - 1.50 (1.84) - - (NH4)5[α-PW11O39{Cu(H2O)}] · 16.5 H2O 58.0 (64.3) n. d. (0.98) - - - n. d. (2.02) - (NH4)5[α-PW11O39{Zn(H2O)}] · 19 H2O 61.9 (63.3) 1.19 (0.97) - - - 5.11 (2.05) (NH4)6[β3-SiW11O39{Co(H2O)}] · 18.5 H2O 58.0 (63.2) - 0.95 (0.88) 1.52 (1.84) - - -

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Table S5: ESI MS analysis of Keggin POTs. Assigned m/z species are listed with the experimental m/z ratios, and the expected values are given in brackets for comparison.

Keggin POT compound assignment m/z

(NH4)5[α-PW11O39{Co(H2O)}] H2[PW11O39Co]3- 912.7 (912.7) H3[PW11O39Co]2- 1369.6 (1369.6)

(NH4)5[α-PW11O39{Ni(H2O)}] H[PW11O39Ni]4- 684.1 (684.2) H2[PW11O39Ni]3- 912.7 (912.7) H3[PW11O39Ni]2- 1369.6 (1369.6)

(NH4)5[α-PW11O39{Cu(H2O)}] H[PW11O39Cu]4- 684.8 (685.5) (NH4)5[α-PW11O39{Zn(H2O)}] H[PW11O39Zn]4- 685.6 (685.8)

(NH4)6[β3-SiW11O39{Co(H2O)}] H2[SiW11O39Co]4- 683.6 (683.8) H3[SiW11O39Co]3- 912.1 (912.1) H4[SiW11O39Co]2- 1368.6 (1368.6)

Table S6: 31P NMR analysis of Keggin POTs. Experimental signals are listed with their ppm shifts, and compared with literature data[9] for the unsubsituted anions [PW11O39]

7- (reference for transition-metal-free POT structure) and [PW12O40]

3- (reference for full P-centered Keggin-POT structure).

Keggin POT compound 31P shifts [ppm] α-[PW11O39{Co(H2O)}]5- +458.4 α-[PW11O39{Ni(H2O)}]5- +472.9 α-[PW11O39{Cu(H2O)}]5- -39.2 α-[PW11O39{Zn(H2O)}]5- -4.8 α-[PW11O39]

7- -11.9 -12.4[9] α-[PW12O40]

3- -14.5 -15.0[9]

3. Crystal structures of proteinase K with Keggin POTs

3.1. Protein crystallization

Crystals with monosubstituted Keggin POTs were obtained by the hanging-drop vapor diffusion technique in 100 mM NaOAc/AcOH pH 5.5 with 0.7-1.2 M (NH4)2SO4 as a precipitant (reservoir solution). Drops were set up from 1 µL of 100 g/L proteinase K stock solution, 0.5 µL of 5 mM Keggin POT stock solution and 1 µL of reservoir solution. 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. The crystals containing the β-isomeric Keggin POT [β3-SiW11O39{Co(H2O)}]6- were grown in 100 mM NaOAc/AcOH pH 4.5 with 0.3-0.7 M (NH4)2SO4 and 0.5 M betaine. The drops were composed of 1 µL of 100 g/L proteinase K stock solution, 1 µL of 20 mM Keggin-POT stock solution and 1 µL of reservoir solution. 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. It was also found as a protein ligand in one crystal structure presented here.

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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. Crystals were measured at beamline ID23-1 at the ESRF in Grenoble, France. Co-crystals with α-[PW11O39Zn]5- were analyzed with a Bruker D8 VENTURE X-ray diffractometer equipped with a multilayer monochromator, a PHOTON II charge-integrating pixel array detector, a Cu Kα Incoatec Microfocus (sealed tube) and a Kryoflex cooling device. For data collection and refinement statistics refer to Table S7.

3.2. X-ray data collection, structure solution and refinement

X-ray data were processed with XDS[10], and the structures were solved by molecular replacement with the PDB protein structure 1ic6 using PHASER[11] within the PHENIX[12] program suite. Structures were refined with phenix.refine (PHENIX) and manually fixed in Coot[13]. Coordinates and restraints files for the α- and β-isomer of the Keggin structure (from PDB entry 4gnl and CCDC entry 1209748, respectively) were created using phenix.elbow (PHENIX) and the POT ligands were modelled in Coot.

Anomalous phasing with tungsten atoms is a great advantage for experimental phasing of unknown structures as the standard synchrotron beams feature wavelengths of about 1 Å, coinciding with high values of the anomalous scattering coefficients for tungsten, and also the less intense Cu K-α irradiation of in-house X-ray sources with 1.54 Å still gives rise to reasonably strong anomalous signal. In order to demonstrate the applicability of the Keggin POTs as experimental phasing tools, the representative proteinase K structure with α-[PW11Co]5- was also solved by the single anomalous dispersion method using EP-PHASER and HySS (PHENIX) for anomalous substructure determination. The determined W positions were used to build in the two Keggin POT positions 1 and 2, and the resulting ligand model was incorporated into a model built by the AutoBuild wizard (PHENIX) from the known protein sequence and manually completed in Coot, followed by refinement with phenix.refine. The obtained structure shows good statistics and proves the applicability of the presented POT clusters as a phasing tool for unknown protein structures. Parameters of data collection are given in Table S7, together with the refinement statistics. The B-factors for the structures with Keggin-POTs α-[PW11Co]5-, α-[PW11Ni]5- and β-[SiW11Co]6- do not significantly differ from the corresponding protein B-factors, supporting the proper refinement of X-ray structures and the covalent bond formation between POT and protein.

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Table S7: X-ray structural analysis, data processing and refinement.  

Keggin POT α-[PW11Co]5- α-[PW11Ni]5- α-[PW11Cu]5- α-[PW11Zn]5- β-[SiW11Co]6- β-[SiW11Co]6- α-[PW11Co]5- Crystal data Space group P43212 P43212 P43212 P43212 P43212 P43212 P43212 a, b, c, [Å] 67.86, 67.86, 102.3 67.96, 67.96,

102.67 67.92, 67.92, 102.18

67.92, 67.92, 102.14

68.42, 68.42, 106.67

67.82, 67.82, 101.66

67.86, 67.86, 102.3

α, β, γ [°] 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 Molecules per asymmetric unit 1 1 1 1 1 1 1 Matthews coefficient [Å3 Da-1] 1.88 1.89 1.88 1.88 1.99 1.86 1.88 Solvent content [%] 0.345 0.349 0.345 0.345 0.382 0.340 0.345 Maximum resolution [Å] 1.1 1.1 1.2 1.35 1.15 1.1 1.1 Data collection and processing Wavelength [Å] 0.97625 0.97625 0.97625 1.542 0.97625 0.97625 0.97625 Resolution limits [Å] 35 - 1.1

(1.139 - 1.1) 30.39 - 1.1 (1.139 - 1.1)

34.99 - 1.2 (1.243 - 1.2)

24.04 - 1.35 (1.398 - 1.35)

35.83 - 1.15 (1.191 - 1.15)

32.17 - 1.1 (1.139 - 1.1)

35 - 1.1 (1.139 - 1.1)

Number of observed reflections 737740 (34845) 620639 (30102) 623728 (61498) 471408 (23114) 654909 (25693) 550251 (26477) 737740 (34845) Number of unique reflections 184016 (17428) 184806 (17305) 142531 (14209) 92760 (8059) 169779 (15345) 181436 (16704) 96998 (9398) Redundancy 4.0 (2.0) 3.4 (1.7) 4.4 (4.3) 5.1 (2.9) 3.9 (1.7) 3.0 (1.6) 7.6 (3.7) Rp.i.m.

[a] 0.09364 (0.4568) 0.1319 (0.1941) 0.06592 (0.1366) 0.01637 (0.112) 0.09024 (2.046) 0.1085 (0.4181) 0.07843 (0.3699) Rmerge.

[b] 0.1743 (0.5654) 0.2197 (0.2462) 0.1241 (0.252) 0.03601 (0.1815) 0.1695 (2.425) 0.1721 (0.4534) 0.2122 (0.6412) CC1/2 0.973 (0.655) 0.93 (0.849) 0.985 (0.934) 0.999 (0.961) 0.982 (0.0893) 0.962 (0.67) 0.979 (0.718) CC* 0.993 (0.89) 0.982 (0.958) 0.996 (0.983) 1 (0.99) 0.996 (0.405) 0.99 (0.896) 0.995 (0.914) Completeness [%] 99.33 (94.19) 99.06 (92.69) 99.79 (99.84) 92.48 (80.41) 98.67 (89.36) 98.62 (90.90) 99.82 (98.34) <I/σI> 5.04 (1.32) 4.83 (2.53) 8.35 (4.71) 27.53 (4.86) 3.63 (0.22) 4.34 (1.27) 6.83 (1.76) Anomalous signal SigAno [c] 1.271 1.398 1.195 1.285 0.923 1.034 1.271 AnomCorr [d] [%] 54 49 47 40 39 30 54 Refinement statistics Resolution [Å] 35 - 1.1 30.39 - 1.1 34.99 - 1.2 24.04 - 1.35 35.83 - 1.15 32.17 - 1.1 35 - 1.1 Reflections used 184012 (17427) 184791 (17301) 142531 (14209) 92750 (8059) 169670 (15349) 181429 (16704) 96995 (9397) Rwork

[e] [%] 14.56 (26.84) 14.73 (17.95) 14.86 (15.95) 14.79 (18.09) 18.88 (42.22) 18.56 (28.35) 12.74 (24.15) Rfree

[f] [%] 15.45 (28.48) 15.85 (18.76) 16.64 (18.33) 18.08 (25.01) 21.82 (42.17) 19.98 (28.67) 14.03 (25.77) Number of water molecules 304 351 339 415 399 335 307 Average B-factor [Å2] 11.03 9.48 12.99 11.50 18.53 12.33 12.16 B-factor : Protein chain 9.39 7.78 9.48 7.11 16.18 9.6 10.43 B-factor : POTs 15.19 8.91 25.21 97.71 23.20 22.17 14.51 B-factor : sulphate (betaine) 12.08 10.99 20.04 9.07 19.91 (16.98) 13.82 13.13 B-factor : water molecules 20.55 19.50 24.67 22.33 28.58 22.77 22.74 Ramachandran plot[g] Most favoured regions [%] 97.09 97.11 97.1 97.11 97.09 97.11 97.09 Additional allowed regions [%] 2.91 2.89 2.9 2.89 2.91 2.89 2.91 Disallowed regions [%] 0 0 0 0 0 0 0 PDB ID 6RUG 6RUH 6RUK 6RUW 6RVE 6RVG 6RUN

 

 

Footnote

[a] Rp.i.m.

reflec

[b] Rmerge

[c] Mean

structclass

[d] Perce

[e] Rwork struct

[f] Rfree iseach

[g] Calcu

3.3. M

Fig. S5: (PDB e[PW11O3

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= Σhkl{1/[N(h

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Electrostatentry: 6RUG

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surface patin white. The

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13  

The two main interaction positions of Keggin-POTs on the surface of proteinase K (cf. Fig. S5) were expected in the vicinity of surface patches with excess positive charge, but finally appeared to be involved in various interaction modes with non-polar and negatively polarized surface patches as well.

Fig. S6: Electron density of covalent bond in position 1. The POT α-[PW11Co]5- (PDB entry: 6RUG) was chosen as a representative compound to illustrate the covalent bond to the aspartate residue D207 with a 2 Fc-Fo electron density map. The electronic interaction of the POT CoII-center with the protein side-chain is clearly evident.

 

Fig. S7: Comprehensive view of Keggin POT interactions with different protein monomers. Distinct protein molecules are represented by different shades of green and the side-chains belonging to the same protein monomer are marked correspondingly with asterisks. One-letter code for amino acids: A, alanine; D, aspartic acid; E, glutamic acid; I, isoleucine; K, lysine; M, methionine; N, asparagine; R, arginine; S, serine; T, threonine; Y, tyrosine. A) Protein interactions around the covalent bond to D207 (position 1). As a representative structure, α-[PW11Ni]5- (PDB entry: 6RUH) was selected. The position of the polyanion is stabilized by main-chain amide and side-chain hydroxyl H-bonding to two more protein chains. B) Protein interactions in the vicinity of the H-bonding network close to S45 (position 2). As a representative structure, α-[PW11Co]5- (PDB entry: 6RUG) was selected. A second proteinase K molecule donates H-bonds to the POT cluster from the opposite side.

14  

The three Keggin POTs substituted by CoII and NiII (α-[PW11Co]5-, α-[PW11Ni]5-, β-[SiW11Co]6-) shared a common position on the proteinase K surface, forming a covalent bond to the aspartate side-chain D207 (position 1). The electron density (Fig. S6) confirms the directed interaction of the POT 3d transition metal site with the carboxylate group in this site. The bridging contacts to three different protein molecules are depicted in Fig. S7A.

All Keggin anions, including also α-[PW11Cu]5- and α-[PW11Zn]5-, were 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 (Fig. S7B).

Fig. S8: Atom numbering of the representative POTs α-[PW11Ni]5- and β-[SiW11Co]6-. The Keggin anions differ in the rotated upper W-W-metal triad, affecting also the positions of the µ2-O atoms 1, 2 and 3 as well as the terminal oxygens 29 and 30 and the aquo-ligand. The presented atom numbering provides the basis for the analysis of protein-POT interactions in Tables S8 and S9. Metal atoms are numbered in light grey and oxygen atoms in black.

15  

Table S8: Stabilizing protein interactions in position 1 (cf. Fig. 1) for two representative structures with the α- (PDB entry: 6RUH) and β-Keggin-POT (PDB entry: 6RVG) isomers, including hydrogen bonds. Equivalent interactions are compared in the same line, in black color for the α-isomeric structure and in dark red color for the β-isomeric structure. It is evident that the protein-POT interactions are highly conserved for both isomers and reflect the shared basic Keggin-POT architecture, with the two isomers differing in one rotated W-W-Co triad. The POT atoms are numbered according to Fig. S8.

POT Coordinating POT oxygen Coordinating protein side-chain Distance [Å] [α-PW11Ni]5-, [β-SiW11Co]6-

Ni-aquo-ligand replaced, O 3 (µ2-bridging)

Asp 207 (Oδ2, covalent bond), Asp 207 (Oδ1, Oδ2, covalent bond)

1.5 (Oδ2-Co), 1.9 (Oδ2-Co), 3.0 (Oδ2-W), 2.8 (Oδ1-W)

[α-PW11Ni]5-, [β-SiW11Co]6- O 3 (µ2-bridging), O 1 (µ2-bridging) Arg 185 (Nε) 3.9, 3.4 - , [β-SiW11Co]6-

- , aquo-ligand

Arg 185 (Nη2) + Asp 207 (Oδ2), Arg 185 (Nη2)

4.5, 2.5

[α-PW11Ni]5-, [β-SiW11Co]6- O 32 (terminal), O 34 (terminal) Tyr 186 (Oη) 1.8, 3.0 [α-PW11Ni]5-, [β-SiW11Co]6- O 30 (terminal), O 33 (terminal) Asp 184 (Oδ2) 3.6, 3.7 - - Asp 184 (Oδ2) + Arg 188 (Nε) 2.8, 2.8 [α-PW11Ni]5-, [β-SiW11Co]6- O 30 (terminal), O 33 (terminal) Arg 188 (Nη2) 2.7, 3.7 [α-PW11Ni]5-, [β-SiW11Co]6- O 4 (µ2-bridging), O 19 (µ2-bridging) Arg 188 (Nη2) 3.6, 3.8

16  

Table S9: Stabilizing protein interactions within the hydrogen-bonding network at position 2 (cf. Fig. 3) for two representative structures with the α- (PDB entry: 6RUH) and β-Keggin-POT (PDB entry: 6RVG) isomers. Equivalent interactions are compared in the same line, in black color for the α-isomeric structure and in dark red color for the β-isomeric structure. Due to anchoring by the aquo-ligand, the protein-POT interactions are again highly conserved for both isomers, even though [β-SiW11Co]6- binds to the protein surface in a different orientation with regard to the α-isomers. The POT atoms are numbered according to Fig. S8.

Ligand Coordinating POT oxygen Coordinating protein side-chain Distance [Å] [α-PW11Ni]5-, [β-SiW11Co]6- Ni-aquo-ligand, Co-aquo-ligand Ser 63 (O) 2.7, 2.7 [β-SiW11Co]6- O 30 (terminal) Ser 63 (Oγ) 3.4 [β-SiW11Co]6- O 29 (terminal) Ser 63 (N) 2.7 [β-SiW11Co]6- O 29 (terminal) Arg 64 (N) 2.8 [α-PW11Ni]5- + H2O 31, [β-SiW11Co]6- + H2O 60 Ni-aquo-ligand, Co-aquo-ligand - 3.2, 3.3 [β-SiW11Co]6- + H2O 60 O 7 (µ2-bridging) - 2.7 [α-PW11Ni]5-, [β-SiW11Co]6- Ni-aquo-ligand, Co-aquo-ligand Ile 42 (O) 2.8, 2.7 [α-PW11Ni]5- O 33 (terminal) Met 55 (N) 2.8 [α-PW11Ni]5-, [β-SiW11Co]6- O 3 (µ2-bridging), O 8 (µ2-bridging) Ala 44 (N) 3.3, 3.5 [α-PW11Ni]5-, [β-SiW11Co]6- O 30 (terminal), O 14 (µ2-bridging) Ser 45 (N) 3.0, 2.9 [α-PW11Ni]5-, [β-SiW11Co]6- O 30 (terminal), O 14 (µ2-bridging) Ser 45 (Oγ) 2.7, 3.3 [β-SiW11Co]6- O 34 (terminal) Ser 45 (Oγ) 3.8 H2O 31 + H2O 93, H2O 60 + H2O 86 - - 2.8, 2.8 H2O 93, H2O 86 - Glu 43 (Oε2) 2.7, 2.5 H2O 93 + H2O 205, H2O 86 + H2O 118 - - 2.2, 2.8 H2O 205 - Arg 64 (Nε) 2.7 H2O 205, H2O 118 - Arg 64 (Nη2), Arg 64 (Nη1) 2.8, 2.9

17  

3.4. Additional positions and conformations of Keggin anions

Some of the polyoxotungstate clusters were also found in additional side positions. In one structure containing β-[SiW11Co]5- (PDB entry: 6RVG), a third copy of the polyanion was identified in a covalent bond with the carboxylate group of D165 (Fig. S9).

Fig. S9: Additional covalent bond formation of β-[SiW11Co]6- with aspartate residue (D165). The metal cluster interacts with three protein molecules, which are depicted in different shades of green. The side-chains belonging to the same protein monomer are marked correspondingly with asterisks. One-letter code for amino acids: D, aspartic acid; G, glycine; K, lysine; R, arginine; T, threonine; Y, tyrosine.

 

Fig. S10: Additional interaction sites for α-[PW11Cu]5- on the surface of proteinase K (PDB entry: 6RUK). One-letter code for amino acids: D, aspartic acid; E, glutamic acid; G, glycine; K, lysine; M, methionine; N, asparagine; Q, glutamine; R, arginine; S, serine; T, threonine; Y, tyrosine. The metal cluster interacts with three protein molecules, which are depicted in different shades of green. The side-chains belonging to the same protein monomer are marked correspondingly with asterisks. The resolved sliding cluster positions are visualized by the use of transparency and different colors for the W atoms and correspond to a pronounced mobility of the POT within the solvent channels of the protein crystal. The copper centers in all shown POT positions face away from the protein surface and do not bind a water ligand, supporting the presumed coordinative saturation of CuII within the POT lacuna and explaining the lack of spatial fixation.

18  

The Cu-containing compound α-[PW11Cu]5- did not participate in any covalent interactions and adopted several resolved positions in addition to position 2 (Fig. S10), illustrating the reduced affinity for this POT cluster to the proteinase K surface.

3.5. Keggin POT occupancies in the crystal structures

An important finding of this study presents a correlation between the substituted transition metal element and the interaction strength with the protein surface, with the Keggin POT occupancy as a suitable measure (Table S10). The observed occupancies for the covalent linkage to side-chain D207 (position 1) are in line with the decreasing hard character of the transition elements in the series Co2+ > Ni2+ > Cu2+ > Zn2+. Even though no covalent bond is formed at the second main interaction site around S45 (position 2), the same trend was observed. This supports the suggested importance of the anchoring function of the aquo-ligand at the transition metal atom for stabilization of the surface interaction at this position. Interestingly, the altered geometry of β-[SiW11Co]6- turned out to be less compatible with this interaction site, adopting two alternative conformations anchored by the shared aquo-ligand position.

The additional and almost equally occupied conformations found for α-[PW11Cu]5- illustrate the weak and transient nature of protein interactions with this cluster. The covalent linkage to carboxylate D165 could only be obtained with the POT β-[SiW11Co]6- under strongly acidic conditions (pH 4.5) in the presence of betaine.

Table S10: Occupancies for various Keggin POT positions in the crystal structures. The occupancies reflect the tendencies of the substituted transition metals to bind to carboxylate side-chains and water molecules. The β-isomeric structure is bound less stably at position 2 than the corresponding α-isomer. The average number of POT molecules per protein molecule in the crystals is obtained as the sum over the mean occupancies for each POT position. Occupancies and B-factors were refined independently.

Keggin POTs Position 1 (D207)

Position 2 (S45)

Further positions POT molecules per protein

α-[PW11Co]5- 0.33 0.88 - 1.21 α-[PW11Ni]5- 0.25 0.62 - 0.87 α-[PW11Cu]5- - 0.36 0.18, 0.17, 0.15/0.14 0.86 α-[PW11Zn]5- - 0.64 - 0.64 β-[SiW11Co]6- 0.37 0.19/0.20 0.36 0.93

19  

4. Structural integrity of proteinase K upon prolonged storage with a Keggin POT

4.1. SDS-PAGE analysis of proteinase K incubated with a Keggin anion

Aside from structural stability of the POT cluster itself, another highly desirable feature of crystallization-suited POTs is their ability to preserve structural integrity of the protein fold. This precludes the application of compounds with undesired proteolytic or other catalytic activities interfering with the native protein structure. To address the question of protein stability in POT-protein mixtures over extended storage times as required for protein crystallization, α-[PW11Ni]5- was used as a representative compound and incubated with proteinase K for four weeks (Fig. S11), revealing full protein integrity during this time as shown by SDS-PAGE analysis.

Fig. S11: SDS-PAGE analysis of long-time stability of proteinase K. The enzyme (1 g/L, 35 µM) was incubated in 100 mM NaOAc pH 5.5 at 20 °C in the presence of 25 µM α-[PW11Ni]5-, using the same protein:POT ratio as in the successful crystallization trials. Aliquots were taken at the indicated time points. Samples were incubated for 10 min with 1 mM phenylmethylsulfonyl fluoride (PMSF) prior to heating in SDS sample buffer in order to block the otherwise exhibited autoproteolytic activity.

Full protein integrity is clearly evident, which sorts out potential Lewis-acid activity of α-[PW11Ni]5- towards the protein backbone, in accordance with the selection of electron-rich transition metals in a low oxidation state that was chosen here for the monosubstituted Keggin anions.

4.2. Enzymatic activity assay in the presence of a Keggin anion

A second important point of interest was the impact of the substituted Keggin POTs on proteinase K activity. The catalytic triad of proteinase K consists of the amino acids Asp39, His69 and Ser224[14] and is not affected by POT binding according to the presented crystal structures. Still, protein activity in solution may be altered by strong interaction with the POTs, which would make them less attractive for protein crystallization as the co-crystallized

20  

state then would hardly reflect the native protein state one is usually interested in. Therefore, activity assays with the chromogenic substrate p-nitrophenylacetate[15] were performed with various concentrations of α-[PW11Ni]5- from 0 – 500 µM (Fig. S12).

 

 

 

Fig. S12: Enzymatic activity assay of proteinase K with α-[PW11Ni]5-. Reactions were set up in 25 mM 3-(N-morpholino)propanesulfonic acid (MOPS) pH 7.4 with an enzyme concentration of 100 µg/mL and a substrate concentration of 2.5 mM p-nitrophenylacetate in a total volume of 1 mL, and reaction progress was monitored for 3 min as the absorbance increase at 425 nm. Linear starting regions of 30 s were evaluated for their slopes and normalized to the activity determined without POT to yield relative activities. These values were fit by a hyperbolic expression derived from Michaelis-Menten kinetics to extract the kinetic parameters Ki and α[16].

The hyperbolic curve fit indicates a slight activity decrease with higher POT concentrations. However, the observed activity decrease in the region around 5 µM POT, reflecting the successful crystallization conditions with a molar POT:protein ratio of about 1.4, is negligible. This supports the finding from the crystal structures that the native protein conformation is basically unchanged by POT binding. Further investigation of the inhibitory capacity of α-[PW11Ni]5- towards proteinase K at much higher POT:protein ratios revealed a mixed inhibition type (α = 0.015, Ki = 2.06 mM) with a strong uncompetitive contribution as observed for Keggin-type inhibitors with the tyrosinase abPPO4 before[16].

Protein deformation can, in principal, result from too high charge density and unspecific aggregation driven by electrostatics, or it can be driven by strong non-polar surface contacts arising from too low charge density as exhibited by very large POM clusters with pronounced chaotropic properties. Thus, proper balance of surface charge density and polarizability is required, as achieved here by the incorporation of divalent transition metal ions. The superchaotropic interactions displayed even by highly charged POMs (up to 5-) constitute a considerable share of their overall surface interactions[17], as has been demonstrated with model membranes[18] and also explains the observation of pronounced interactions with uncharged molecules such as polyethylene glycol[19]. Notably, both main Keggin POT

21  

interaction sites on proteinase K were identified as cavities with inhomogeneous surface polarity, featuring charged, neutral and even hydrophobic patches (cf. Fig. S5), in line with the versatile surface interactions exhibited by POTs.

5. CCDC data base survey on monosubstituted Keggin POTs with ligands at the transition metal center

In order to integrate the presented observations of transition metal coordination chemistry into a broader view on existing structural data, an extensive data base search of the Cambridge Crystallographic Data Centre (database of metal-organic and organic structures, CCDC, https://www.ccdc.cam.ac.uk) was performed, using the search expressions “Keggin”, “undeca-tungst” and “undecatungst” to obtain hits only for POT anions. Only monosubstituted Keggin POTs with a single transition metal bound to at least four oxygen atoms of the POT lacuna were further investigated. Excluded from this survey were all those clusters with: main group elements or lanthanides in the lacunary site, more than one metal bound to the lacunary site, less than four oxygen atoms from the lacuna binding to the transition metal, or with a vanadium or molybdenum atom as the substituting transition metal. The latter two elements exclusively carry inert terminal oxo ligands, reflecting their nature as addenda atoms constituting POM frameworks, and were therefore not of interest for this survey.

The resulting 100 entries (as of May 2019) were analyzed for the coordination geometry of the incorporated transition metal, the type of ligand attached to the transition metal and for further binding interactions of bridging or terminal oxygen atoms, and finally arranged into Table S11.

Transition metals of nearly every auxiliary group of the periodic table were incorporated to monolacunary Keggin POTs and crystallized. ScIII-, YIII- and CrIII-substituted Keggin POTs exist, but were not found in the CCDC. POT structures in the solid state generally have to be interpreted with precaution. It must be emphasized that the crystallization conditions are usually not comparable to biological conditions regarding extreme pH values, high metal concentrations and organic solvents. Before this background, the high number of polymeric chains (linked by bonds between the transition metal and terminal oxygens or additional metal complexes) in the presented data should be seen as the result of strong dehydration during the crystallization process and not be confused with a realistic compound state in more dilute and bio-compatible aqueous solution. Still, focussing rather on the local coordination geometry of the transition metals than the macroscopic assemblies formed, the following general findings were revealed through the survey:

1. The early transition metals (TiIV, NbV) tend to bear terminal oxo ligands showing pronounced pH-dependent nucleophilicity, leading to dimerization and transmetallation reactions, in contrast to the chemically inert terminal oxo groups at the addenda atoms VIV/V, MoVI or WVI. As their increased size compromises proper insertion into the rigid POT lacuna, the oxophilic higher homologues of TiIV, ZrIV and HfIV, can be found in extended coordination modes involving two POT units, exceeding the octahedral geometry.

22  

2. The higher homologues of first row transition elements with increased ionic radii prefer soft ligand atoms for binding (e.g. Ru-S, Ru-N, Rh-C) over hard oxygen atoms, following the HSAB concept.

3. The pentadentate Keggin POT lacuna predefines the octahedral coordination geometry for most transition metals, which is completed by a sixth terminal ligand. However, elements of group XIa (like CuII) and XIIa (like ZnII) can also adopt a square pyramidal configuration without terminal ligand due to their electronic saturation, which is in perfect agreement with the POT crystal structures presented in this study.

4. For better steric accessibility, the terminal oxo-groups Ot are more often involved in bridging contacts than the µ2-O atoms. Still, those µ2-O bridging a W-metal unit are found more frequently in interactions with external metals than those connecting two W atoms, in accordance with the theoretical considerations pointed out before.

Table S11: CCDC survey on Keggin POTs monosubstituted by transition metals with ligands. All the 100 hits (as of 11.05.2019) represent the α-Keggin isomer. The POT structures relevant for this study, with or without aquo-ligands, are highlighted in colors. The transition metal and the ligand atom binding to it are depicted in bold letters. For clarity, only the anionic POT unit is considered in detail. Abbreviations: CTP, capped trigonal prismatic; O, octahedral; L, neutral ligand which is not connected to the substituting transition metal center, but to an external metal ion; PB, pentagonal bipyramidal; SA, square antiprismatic; SP, square pyramidal; TM, transition metal.

23  

PSE group of TM

Formula of Keggin POT anion Coordination

geometry of TM Ligand at transition metal Keggin µ2-O binds to Keggin Ot binds to CCDC entry Reference

IVa

[PW11O39TiIVO]5- O oxo - - 1298665, 906701 [20], [21] [(PW11O39TiIV)2(µ2-O)]8- O titanyl - TiIV of another POT unit 906699 [21] [PW11O39TiIV(η2-O2)]

5- CTP peroxo - - 1298666 [20] ([SiW11O39ZrIV(µ2-OH)]5-)2 PB hydroxo - - 1526102 [22] [(PW11O39)2ZrIV]10- SA lacuna of other POT unit - - 702812, 702813 [23] ([PW11O39HfIV(OH2)(µ2-OH)]4-)2 CTP aquo, hydroxo - - 784218 [24] H3[PW11O39HfIV(N4C40H24N4)]

2- SA porphyrinato - - 717412 [25]

Va

[PW11O39NbVO]4- O oxo - - 1587892 [26] [PW11O39NbV(OGe(CH3)3)]

3- O trimethylgermanyloxo - - 1242959 [27] [(PW11O39NbVO)2ZrIV(C5H5)2]

6- O zirconocenyl dioxo - - 135355 [28] [PW11O39NbV(OPbIIL2(µ2-L3)PbIIL3)]n O (dimethylformamid) plumboxo - external PbII 1859950 [29]

VIIa

[SiW11O39MnII(OH2)]6- O aquo - - 606862, 1141681 [30], [31]

[SiW11O39MnII(NC3H4N)]6- O imidazole - - 606862 [30] ([PW11O39MnII]5-)n O Ot of next POT unit - MnII of next POT unit 1308684, 1308685 [32], [33] (H[BW11O39MnII]6-)n O Ot of next POT unit - MnII of next POT unit 763016 [34] [PW11O39ReVO]4- O oxo - - 851154 [35]

VIIIa

[SiW11O39FeIII(OH2)]5- O aquo - - 734482 [36]

[PW11O39FeIII((µ2-OH)FeIIIL2)]2- O hydroxo external FeIII - 649965 [37]

[SiW11O39RuIII(OH2)]5- O aquo - - 997028, 997029 [38]

[PW11O39RuII(S(CH3)2O)]5- O S-dimethylsulfoxide - - 824483 [39] [GeW11O39RuIII(S(CH3)2O)]5- O S-dimethylsulfoxide - - 898463 [40] [SiW11O39RuII(S(CH3)2O)]6- O S-dimethylsulfoxide - - 606353 [41] [GeW11O39RuII(S(CH3)2O)]6- O S-dimethylsulfoxide - - 953950 [42] [SiW11O39RuIII(NC6H4O2)]

5- O N-pyridine-4-carboxylato - - 847809 [43] [SiW11O39RuIII(NC7H8NO)]5- O N-4-pyridylacetamide - - 847811 [43] [SiW11O39RuIII(NC10H8N)]5- O 4,4’-bipyridine - - 847810 [43]

IXa

[PW11O39CoII(OH2)]5- O aquo - -

1059479, 1059480, 692452

[44], [45]

([SiW11O39CoII((µ2-OH)CoIIIL2)]4-)n O hydroxo external CoII - 634947 [46]

[PW11O39CoII(NC3H4N)]5- O imidazole - - 971272 [47] H3[PW11O39CoII(NC5H5)]

2- O pyridine - - 1483189 [48] [SiW11O39CoII(NC10H8N)]6- O 4,4’-bipyridine - - 689779 [49] [PW11O39CoII(NC16H11NBr)]5- O (4-bromophenyl)-2-(4-pyridinyl)pyridine external AgI external AgI 238955 [50] [PW11O39CoII(NC16H12N)]5- O 5-phenyl-2-(4-pyridinyl)pyridine - - 232770 [51]

([PW11O39CoII]5-)n O Ot of next POT unit - CoII of next POT unit 153421, 1273306, 295616

[52], [53], [54], [55]

([AsW11O39CoII]5-)n O Ot of next POT unit - CoII of next POT unit 1273307, 1532827 [53], [56] ([SiW11O39CoII]6-)n O Ot of next POT unit - CoII of next POT unit 948815, 642470 [54], [57]

([SiW11O39CoII]6-)n O Ot of next POT unit - CoII of next POT unit,

external CoII 785385 [58]

[SiW11O39RhII(CH2CO2H)]6- O carboxymethyl - - 105680 [59]

24  

Xa

[PW11O39NiII(OH2)]5- O aquo - - 1258198, 1495993 [33], [60]

[SiW11O39NiII(OH2)]6- O aquo - - 606861, 675954 [30], [61]

[SiW11O39NiII(NC3H4N)]6- O imidazole - - 606861 [30] H2[SiW11O39NiII(NC4H6N)]4- O 2-methyl-1H-imidazole - external NiIII 1028317 [62] [PW11O39NiII(NC10H9N)]4- O 4,4’-bipyridinium - - 690126, 757166 [63], [64] ([SiW11O39NiII(NC10H8NNiIIL4)]

2-)n O 4,4’-bipyridine - external NiII 279377 [65] ([SiW11O39NiII]6-)n O Ot of next POT unit - NiII of next POT unit 206688 [66] [SiW11O39PtIV(OH2)]

4- O aquo - - 1050458 [67]

XIa

[PW11O39CuII]5- SP - - - 824098, 798572, 696797, 696798

[68], [69], [70]

[PW11O39CuII(OH2)]5- O aquo - - 734457, 686647 [71], [72]

[SiW11O39CuII(OH2]6- O aquo - external CuII 294601 [73]

[PW11O39CuII(OPO3)]8- O phosphato external CuII external CuII 272557, 633134

[74], [75], [76]

[PW11O39CuII(NC10H8N)]5- O 4,4’-bipyridine external CuII external CuII 272558 [74] [SiW11O39CuII(µ2-ClCuIIL2)]

4- O chloro - - 656525 [77] ([SiW11O39CuII]2(OH2))

12- O aquo, Ot of another POT unit - CuII of POT 242455 [78] ([SiW11O39CuII]2(µ2-OH2))

12- O aquo - external CuII 267526 [79]

([PW11O39CuII]5-)n O Ot of next POT unit - CuII of next POT unit 948814, 798988, 981289, 981291

[54], [80], [81]

([PW11O39CuII]5-)n O Ot of next POT unit external AgI CuII of next POT unit,

external AgI 825927 [82]

([SiW11O39CuII]6-)n O Ot of next POT unit - CuII of next POT unit 625858, 656530, 234031, 675953

[61], [77], [83], [84]

([SiW11O39CuII]6-)n O Ot of next POT unit external CuII CuII of next POT unit,

external CuII 625860 [83]

([SiW11O39CuII]6-)n O Ot of next POT unit - CuII of next POT unit,

external CuII

656527, 694473, 694474, 882718, 1016093

[77], [85], [86], [87]

([GeW11O39CuII]6-)n O Ot of next POT unit - CuII of next POT unit 656529 [77]

([GeW11O39CuII]6-)n O Ot of next POT unit - CuII of next POT unit,

external CuII 656528 [77]

([SiW11O39CuI]7-)n O Ot of next POT unit - CuI of next POT unit 625859 [83] ([PW11O39CuII(µ2-ClCuIIL2)]

4-)n O chloro external CuII external CuII 215963, 656526 [77], [88] [PW11O39AgI(OH2)]

6- O aquo - - 918151 [89] [PW11O39AgI((µ2-OH)2AgI)]7- O hydroxo external AgI external AgI 912981 [90] ([PW11O39AgI]6-)n SA 4 µ2-O of next POT unit AgI of next POT unit - 605319 [91]

XIIa

([PW11O39ZnII]5-)n O Ot of next POT unit - ZnII of next POT unit 690127 [63] H3[PW11O39ZnII(NC5H5)]

2- O pyridine - - 1483188 [48] [SiW11O39ZnII(NC12H10N2)]

5- O 4-(1H-benzimidazol-2-yl)pyridinium - - 955862 [92] [PW11O39CdII]5- SP - - - 796142 [93]

25  

Table S12: Transition metal-oxygen donor atom bond lengths of CoII- and NiII-substituted Keggin-POTs. The respective compounds from Table S11 were analyzed for their metal-ligand distances to derive an average expected bond length for the covalent bond with aspartate D207 of proteinase K.

CCDC number metal-O distance Average bond length 1059479 1.71, 1.85, 1.78 1.78 1059480 2.08, 1.85, 1.79, 1.73 1.86 692452 1.82 1.82 634947 1.97 1.97 1258198 1.82, 1.82 1.82 1495993 1.97 1.97 606861 1.76 1.76 675954 1.99 1.99

The bond length variation between the oxygen-ligand and the transition metal (Table S12) covers a range of 1.7-2.1 Å. The experimental range from the crystal structures presented in this study (1.5-1.9 Å) is slightly shortened, but still chemically plausible, as bonds to metal centers are generally more flexible in nature than those between non-metals. The findings of the presented survey provide explanations for the observed covalent binding interactions between proteinase K and the tested substituted POT anions. However, they indicate that softer and stronger σ-donor ligands such as histidine or cysteine, which are unavailable on the surface of proteinase K, might support binding of Cu- and Zn-substituted POT clusters as well, opening potential applications of these anions for crystallization of different proteins under different conditions.

6. Acknowledgements

We would also like to thank our colleagues Dipl.-Ing. Ioannis Kampatsikas and Lukáš Krivosudský, PhD for their support during the beamtime. The authors are grateful to Emir Al-Sayed, MSc, Elias Tanuhadi, MSc, Mag. Anatolie Dobrov and Lukáš Krivosudský, PhD for providing the Keggin POTs under study. We also express our thanks to Ass.-Prof. Dr. P. Unfried (Institut für Anorganische Chemie, Universität Wien) for TGA, to RNDr Marek Bujdoš, PhD (Institute of Laboratory Research on Geomaterials, Comenius University, Bratislava), for elemental analysis, to Anna Fabisikova, MSc (Massenspektrometriezentrum, Universität Wien), for ESI-MS measurements and to Ricarda Bugl (NMR Zentrum, Universität Wien) for NMR measurements. Finally, we thank Dr. Tim Grüne and Ing. Alexander Roller, Dipl.-Wirtschaftsing. (FH), (Zentrum für Röntgenstrukturanalyse, Universität Wien) for support with the in-house X-ray measurement, and Nadiia Gumerova, PhD, for proof-reading this manuscript.

7. References  [1] J. C. Bailar, in Inorganic Syntheses, ed. H. S. Booth, The McGraw-Hill Book

Company, Inc., New York, 1939, vol. 1, pp. 132–133.

26  

 [2] T. J. R. Weakley and S. A. Malik, J. Inorg. Nucl. Chem., 1967, 29, 2935–2944, DOI:

10.1016/0022-1902(67)80126-X.

[3] A. Tézé, G. Hervé, R. G. Finke and D. K. Lyon, in Inorganic Syntheses, ed. A. P. Ginsberg, John Wiley & Sons, Inc., Hoboken, 1990, vol. 27, pp. 85–96.

[4] L. C. W. Baker and J. S. Figgis, J. Am. Chem. Soc., 1970, 92, 3794-3797, DOI: 10.1021/ja00715a047.

[5] W. Guan, L.-K. Yan, Z.-M. Su, E.-B. Wang and X.-H. Wang, Int. J. Quantum Chem., 2006, 106, 1860–1864, DOI: 10.1002/qua.20965.

[6] X. Lopéz, I. A. Weinstock, C. Bo, J. P. Sarasa and J. M. Poblet, Inorg. Chem., 2006, 45, 6467-6473, DOI: 10.1021/ic060112c.

[7] D. Karimian, B. Yadollahi and V. Mirkhani, Microporous Mesoporous Mater., 2017, 247, 23-30, DOI: 10.1016/j.micromeso.2017.03.048.

[8] T. L. Jorris, M. Kozik, N. Casan-Pastor, P. J. Domailles, R. G. Finke, W. K. Miller and L. C. W. Baker, J. Am. Chem. Soc., 1987, 109, 7402-7408, DOI: 10.1021/ja00258a025.

[9] R. I. Maksimovskaya and G. M. Maksimov, Coord. Chem. Rev., 2019, 385, 81-99,

DOI: 10.1016/j.ccr.2019.01.014.

[10] W. Kabsch, Acta Cryst. D, 2010, 66, 125–132, DOI: 10.1107/S0907444909047337.

[11] A. J. McCoy, R. W. Grosse-Kunstleve, P. D. Adams, M. D. Winn, L. C. Storoni and R. J. Read, J. Appl. Crystallogr., 2007, 40, 658–674, DOI: 10.1107/S0021889807021206.

[12] P. D. Adams, P. V. Afonine, G. Bunkóczi, V. B. Chen, I. W. Davis, N. Echols, J. J. Headd, L.-W. Hung, G. J. Kapral, R. W. Grosse-Kunstleve , A. J. McCoy, N. W. Moriarty, R. Oeffner, R. J. Read, D. C. Richardson, J. S. Richardson, T. C. Terwilliger and P. H. Zwart, Acta Cryst. D, 2010, 66, 213–221, DOI: 10.1107/S0907444909052925.

[13] P. Emsley and K. Cowtan, Acta Cryst. D, 2004, 60, 2126–2132, DOI: 10.1107/S0907444904019158.

[14] C. Betzel, S. Gourinath, P. Kumar, P. Kaur, M. Perbandt, S. Eschenburg and T. P. Singh, Biochem., 2001, 40, 3080-3088, DOI: 0.1021/bi002538n.

[15] L. A. Stone, G. S. Jackson, J. Collinge, J. D. F. Wadsworth and A. R. Clarke, Biochem., 2007, 46, 245-252, DOI: 10.1021/bi061646s.

[16] J. Breibeck, N. I. Gumerova, B. B. Boesen, M. Galanski and A. Rompel, Sci. Rep., 2019, 9, 5183, DOI: 10.1038/s41598-019-41261-7.

[17] K. Assaf and W. Nau, Angew. Chem. Int. Ed., 2018, 57, 13968-13981, DOI: 10.1002/anie.201804597.

27  

  [18] D. Kobayashi, H. Nakahara, O. Shibata, K. Unoura and H. Nabika, J. Phys. Chem. C,

2017, 121, 12895-12902, DOI: 10.1021/acs.jpcc.7b01774. [19] T. Buchecker, X. Le Goff, B. Naskar, A. Pfitzner, O. Diat and P. Bauduin, Chem. Eur.

J., 2017, 23, 8434-8442, DOI: 10.1002/chem.201700044.

[20] Y. Toshihiro, O. Tomoji and M. Shigeyuki, Bull. Chem. Soc. Jpn., 1992, 65, 1453-1459, DOI: 10.1246/bcsj.65.1453.

[21] Y. Matsuki, Y. Mouri, Y. Sakai, S. Matsunaga and K. Nomiya, Eur. J. Inorg. Chem., 2013, 2013, 1754-1761, DOI: 10.1002/ejic.201201290.

[22] T. Yang, X. X. Li and S. T. Zheng, Jiegou Huaxue, 2017, 36, 1729, DOI: 10.14102/j.cnki.0254-5861.2011-1578.

[23] L. Cai, Y. Li, C. Yu, H. Ji, Y. Liu and S. Liu, Inorg. Chim. Acta, 2009, 362, 2895-2899, DOI: 10.1016/j.ica.2009.01.013.

[24] M. N. Sokolov, N. V. Izarova, E. V. Peresypkina, A. V. Virovets and V. P. Fedin, Russ. Chem. Bull., 2009, 58, 507-512, DOI: 10.1007/s11172-009-0048-9.

[25] A. Falber, B. P. Burton‐Pye, I. Radivojevic, L. Todaro, R. Saleh, L. C. Francesconi and C. M. Drain, Eur. J. Inorg. Chem., 2009, 2009, 2459–2466, DOI: 10.1002/ejic.200900284.

[26] A. A. Shmakova, M. M. Akhmetova, V. V. Volchek, T. E. Romanova, I. Korolkov, D. G. Sheven, S. A. Adonin, P. A. Abramov, M. N. Sokolov, New J. Chem., 2018, 42, 7940-7948, DOI: 10.1039/C7NJ04702A.

[27] E. V. Radkov, R. H. Beer, Inorg. Chim. Acta, 2000, 297, 191-198, DOI: 10.1016/S0020-1693(99)00360-6.

[28] E. V. Radkov, V. G. Young Jr. and R. H. Beer, J. Am. Chem. Soc., 1999, 121, 8953-8954, DOI: 10.1021/ja9905039.

[29] E. P. Bushmeleva, N. B. Kompankov, R. R. Shiriyazdanov, A. R. Karimova, P. A. Abramov and M. N. Sokolov, Inorg. Chem. Comm., 2018, 98, 180-183, DOI: 10.1016/j.inoche.2018.10.026.

[30] H. Liu, C. J. Gómez-García, J. Peng, J. Sha, Y. Li and Y. Yan, Dalton Trans., 2008, 0, 6211-6218, DOI: 10.1039/B805714A.

[31] V. S. Sergienko and M. A. Porai-Koshits, Zhurnal Neorganicheskoi Khimii, 1985, 30, 2282-2285.

[32] J. R. Galán-Mascarós, C. Gimenéz-Saiz, S. Triki, C. J. Gómez-García, E. Coronado and L. Ouahab, Angew. Chem. Int. Ed., 1995, 34, 1460-1462, DOI: 10.1002/anie.199514601.

[33] E. Coronado, J. R. Galán-Mascarós, C. Gimenéz-Saiz, C. J. Gómez-García and S. Triki, J. Am. Chem. Soc., 1998, 19, 4671–4681, DOI: 10.1021/ja973856l.

[34] H. An, H. Zheng, T. Xu, C. He, J. Wang, P. Wu and C. Duan, Z. Anorg. Allg. Chem., 2010, 636, 2016-2021, DOI: 10.1002/zaac.201000068.

28  

 [35] C. N. Kato, Y. Makino, M. Yamasaki, Y. Kataoka, Y. Kitagawa and M. Okumura,

Advances in Crystallization Processes, 2012, 23, 593-610, DOI: 10.5772/2672.

[36] W. L. Chen, B. W. Chen, H. Q. Tan, Y. G. Li, Y. H. Wang and E. B. Wang, J. Solid State Chem., 2010, 183, 310-321, DOI: 10.1016/j.jssc.2009.11.016.

[37] C. Pichon, A. Dolbecq, P. Mialane, J. Marrot, E. Rivière, M. Goral, M. Zynek, T. McCormac, S. A. Borshch, E. Zueva and F. Sécheresse, Chem. Eur. J., 2008, 14, 3189-3199, DOI: 10.1002/chem.200700896.

[38] D. Shi, C. He, B. Qi, C. Chen, J. Niu and C. Duan, Chem. Sci., 2015, 6, 1035-1042, DOI: 10.1039/C4SC02362E.

[39] M. Sadakane, N. Rinn, S. Moroi, H. Kitatomi, T. Ozeki, M. Kurasawa, M. Itakura, S. Hayakawa, K. Kato, M. Miyamoto, S. Ogo, Y. Ide and T. Sano, Z. Anorg. Allg. Chem., 2011, 637, 1467–1474, DOI: 10.1002/zaac.201100213.

[40] S. Ogo, N. Shimizu, T. Ozeki, Y. Kobayashi, Y. Ide, T. Sano and M. Sadakane, Dalton Trans., 2013, 42, 2540-2545, DOI: 10.1039/c2dt32038j.

[41] M. Sadakane, D. Tsukuma, M. H. Dickman, B. Bassil, U. Kortz, M. Higashijima and W. Ueda, Dalton Trans., 2006, 0, 4271-4276, DOI: 10.1039/B606266K.

[42] N. Shimizu, T. Ozeki, H. Shikama, T. Sano, M. Sadakane, J. Clust. Sci., 2014, 25, 755-770, DOI: 10.1007/s10876-013-0641-9.

[43] M. Sadakane, S. Moroi, Y. Iimuro, N. Izarova, U. Kortz, S. Hayakawa, K. Kato, S. Ogo, Y. Ide, W. Ueda and T. Sano, Chem. Asian J., 2012, 7, 1331-1339, DOI: 10.1002/asia.201100853.

[44] F. M. Santos, P. Brandão, V. Félix, H. I. S. Nogueira and A. M. V. Cavaleiro, Polyhedron, 2015, 101, 109-117, DOI: 10.1016/j.poly.2015.07.032.

[45] B. Li, S. T. Zheng and G. Y. Yang, Jiegou Huaxue, 2009, 28, 531.

[46] Y. Wang, Z. Zhang, E. Wang, Y. Qi and S. Chang, Aust. J. Chem., 2008, 61, 874-880, DOI: 10.1071/CH08171.

[47] W. Wang, Y. Qiu and L. Xu, J. Coord. Chem., 2014, 67, 797-806, DOI: 10.1080/00958972.2014.908464.

[48] H. Miao, Y. Dong, Z. Chen, X. He, G. Hu and Y. Xu, Dalton Trans., 2016, 45, 12717-12722, DOI: 10.1039/c6dt02320g.

[49] H. Liu, C. J. Gómez-García, J. Peng, J. Sha, L. Wang and Y. Yan, Inorg. Chim. Acta, 2009, 362, 1957-1962, DOI: 10.1016/j.ica.2008.09.014.

[50] Z. Han, Y. Zhao, J. Peng, H. Ma, Q. Liu, E. Wang, N. Hu and H. Jia, Eur. J. Inorg. Chem., 2005, 2005, 264-271, DOI: 10.1002/ejic.200400471.

[51] Z. Han, Y. Zhao, J. Peng, H. Ma, Q. Liu and E. Wang, J. Mol. Struct., 2005, 738, 1-7, DOI: 10.1016/j.molstruc.2004.07.004.

[52] B. Yan, Y. Xu, X. Bu, N. K. Goh, L. S. Chia and G. D. Stucky, Dalton Trans., 2001, 0, 2009-2014, DOI: 10.1039/B103024H.

29  

 [53] H. T. Evans Jun., T. J. R. Weakley and G. B. Jameson, J. Chem. Soc., Dalton Trans.,

1996, 0, 2537-2540, DOI: 10.1039/DT9960002537.

[54] X. Zhao, J. Yan, X. Xue, Z. Han, S. Cui, L. Zong, D. Zheng, C. Shen, H. Yu and X. Zhai, Inorg. Chim. Acta, 2014, 414, 46-52, DOI: 10.1016/j.ica.2014.01.033.

[55] J. P. Wang, Y. Shen and J. Niu, J. Coord. Chem., 2007, 60, 1183-1190, DOI: 10.1080/00958970601026699.

[56] C. Wu, J. Lv, K. Yu, H. Zhang, C. Wang, C. Wang and B. Zhou, J. Coord. Chem., 2017, 70, 1862-1871, DOI: 10.1080/00958972.2017.1327046.

[57] X. L. Wang, H. Y. Lin, G. C. Liu, B. K. Chen and Y. F. Bi, Chem. Res. Chinese U., 2008, 24, 129-134, DOI: 10.1016/S1005-9040(08)60027-6.

[58] H. Wang, W. You, B. Meng, X. Sun, H. Cheng and W. Shan, J. Clust. Sci., 2010, 21, 857-865, DOI: 10.1007/s10876-010-0346-2.

[59] X. Wei, M. H. Dickman and M. T. Pope, J. Am. Chem. Soc., 1998, 120, 10254-10255, DOI: 10.1021/ja980993p.

[60] C. J. Wang, S. Yao, Y. Z. Chen, Z. M. Zhang and E. B. Wang, RSC Adv., 2016, 6, 99010-99015, DOI: 10.1039/c6ra19257b.

[61] D. J. Li, S. T. Zheng, G. Y. Yang, Jiegou Huaxue, 2009, 28, 655.

[62] Y. Q. Jiao, M. T. Li, C. Qin and Z. M. Su, Cryst. Eng. Comm., 2017, 19, 1721-1724, DOI: 10.1039/C6CE02420C.

[63] L. Chen, F. L. Jiang, N. Li, W. T. Xu and M. C. Hong, J. Clust. Sci., 2008, 19, 591-600, DOI: 10.1007/s10876-008-0199-0.

[64] Y. Wang, F. Q. Wu, L. Ye, T. G. Wang, G. W. Wang, S. Y. Shi, L. N. Xiao, X. B. Cui and J. Q. Xu, Inorg. Chem. Comm., 2010, 13, 703-705, DOI: 10.1016/j.inoche.2010.03.023.

[65] P. Q. Zheng, Y. P. Ren, L. S. Long, Huang R. B. and L. S. Zheng, Inorg. Chem., 2005, 44, 1190-1192, DOI: 10.1021/ic048708s.

[66] J. Y. Niu, Z. L. Wang and J. P. Wang, J. Solid State. Chem., 2004, 177, 3411-3417, DOI: 10.1016/j.jssc.2004.06.001.

[67] P. Klonowski, J. C. Goloboy, F. J. Uribe-Romo, F. Sun, L. Zhu, F. Gándara, C. Wills, R. J. Errington, O. M. Yaghi and W. G. Klemperer, Inorg. Chem., 2014, 53, 13239-13246, DOI: 10.1021/ic502617k.

[68] Y. Wang, Y. Peng, L. N. Xiao, Y. Y. Hu, L. M. Wang, Z. M. Gao, T. G. Wang, F. Q. Wu, X. B. Cui and J Q. Xu, CrystEngComm, 2012, 14, 1049-1056, DOI: 10.1039/C1CE05633F.

[69] J. Song, Z. Luo, D. K. Britt, H. Furukawa, O. M. Yaghi, K. I. Hardcastle and C. L. Hill, J. Am. Chem. Soc., 2011, 133, 16839-16846, DOI: 10.1021/ja203695h.

[70] B. Li, D. Zhao and G. Y. Yang, J. Clust. Sci., 2009, 20, 629-639, DOI: 10.1007/s10876-009-0264-3.

30  

 [71] K. Fukaya, A. Srifa, E. Isikawa and H. Naruke, J. Mol. Struct., 2010, 979, 221-226,

DOI: 10.1016/j.molstruc.2010.06.030.

[72] J. W. Zhao, J. Zhang, S. T. Zheng and G. Y. Yang, Jiegou Huaxue, 2008, 27, 933.

[73] S. Reinoso, P. Vitoria, J. M. Gutiérrez-Zorrilla, L. Lezama, L. San Felices and J. I. Beitia, Inorg. Chem., 2005, 44, 9731-9742, DOI: 10.1021/ic051134m.

[74] L. Lisnard, A. Dolbecq, P. Mialane, J. Marrot, E. Codjovi and F. Sécheresse, Dalton Trans., 2005, 0, 3913-3920, DOI: 10.1039/B507124K.

[75] X. Wang, X. Lu, P. Li, X. Pei and C. Ye, J. Coord. Chem., 2008, 61, 3753-3762, DOI: 10.1080/00958970802120192.

[76] X. Lu, X. Shi, Y. Bi, C. Yu, Y. Chen and Z. Chi, Eur. J. Inorg. Chem., 2006, 2006, 5267-5276, DOI: 10.1002/ejic.200900560.

[77] J. W. Zhao, S. T. Zheng and G. Y. Yang, J. Solid State Chem., 2008, 181, 2205-2216, DOI: 10.1016/j.jssc.2008.04.042.

[78] S. Reinoso, P. Vitoria, L. San Felices, L. Lezama and J. M. Gutiérrez-Zorrilla, Chem. Eur. J., 2005, 11, 1538-1548, DOI: 10.1002/chem.200400762.

[79] S. Reinoso, P. Vitoria, L. San Felices, L. Lezama and J. M. Gutiérrez-Zorrilla, Inorg. Chem., 2006, 45, 108-118, DOI: 10.1021/ic051322c.

[80] H. Pang, H. Man, Y. Yu, M. Yang, Y. Xun and B. Liu, J. Solid State Chem., 2012, 186, 23-28, DOI: 10.1016/j.jssc.2011.11.032.

[81] M. Emirdag-Eanesa, B. Önen and C. D. McMillen, Inorg. Chim. Acta, 2015, 427, 219-225, DOI: 10.1016/j.ica.2014.12.018.

[82] C. J. Zhang, H. J. Pang, Q. Tang and Y. G. Chen, Dalton Trans., 2012, 41, 9365-9372, DOI: 10.1039/C2DT30152K.

[83] L. San Felices, P. Vitoria, J. M. Gutiérrez-Zorrilla, L. Lezama and S. Reinoso, Inorg. Chem., 2006, 45, 7748–7757, DOI: 10.1021/ic060762g.

[84] L. San Felices, P. Vitoria, J. M. Gutiérrez-Zorrilla, S. Reinoso, J. Etxebarria and L. Lezama, Chem. Eur. J., 2004, 10, 5138-5146, DOI: 10.1002/chem.200400279.

[85] H. Fu, Y. G. Li, Y. H. Wang, X. L. Wang, X. J. Feng and E. B. Wang, Inorg. Chim. Acta, 2009, 362, 3231-3237, DOI: 10.1016/j.ica.2009.02.029.

[86] L. Chen, J. Luo, J. Yuan and H. Y. Li, Huaxue Yanjiu, 2012, 23, 13.

[87] J. Luo, H. Li, X. Chen, J. Ma, L. Chen and J. Zhao, Inorg. Chem. Comm., 2014, 50, 19-23, DOI: 10.1016/j.inoche.2014.10.003.

[88] L. Lisnard, A. Dolbecq, P. Mialane, J. Marrot and F. Sécheresse, Inorg. Chim. Acta, 2004, 357, 845-852, DOI: 10.1016/j.ica.2003.09.007.

[89] Y. Wang, L. M. Wang, Y. Y. Hu, L. N. Xia, D. C. Zhao, H. Y. Guo, Z. M. Gao, T. G. Wang, X. B. Cui and J. Q. Xu, Polyhedron, 2014, 83, 2-9, DOI: 10.1016/j.poly.2014.03.030.

31  

 [90] Z. Y. Shi, J. Peng, Y. G. Li, Z. Y. Zhang, X. Yu, K. Alimaje and X. Wang, Cryst. Eng.

Comm., 2013, 15, 7583-7588, DOI: 10.1039/C3CE41312H.

[91] H. I. S. Nogueira, F. A. Almeida Paz, P. A. F. Teixeira and J. Klinowski, Chem. Comm., 2006, 0, 2953-2955, DOI: 10.1039/B605973B.

[92] T. Lu, X. Xu, H. Li, Z. Li, X. Zhang, J. Ou and M. Mei, Dalton Trans., 2015, 44, 2267-2275, DOI: 10.1039/C4DT03092C.

[93] J. Q. Sha, L. Y. Liang, X. Li, Y. Zhang, H. Yan and G. Chen, Polyhedron, 2011, 30, 1657-1662, DOI: 10.1016/j.poly.2011.03.044.