Mehdi Sahihi
email: m.sahihi@chem.ui.ac.ir

Yousef Ghayeb

Binding of biguanides to β-lactoglobulin: molecular-docking and molecular dynamics simulation studies

Keywords: biguanides, β-lactoglobulin, binding affinity, molecular-docking, molecular dynamics simulation

Introduction

Two types of diabetes mellitus are recognised: type I or insulin-dependent diabetes and type II or insulinindependent diabetes. Biguanides are a class of drugs derived from biguanide and they are the most commonly used drugs for diabetes mellitus or pre-diabetes treatment. In addition, their ability to inhibit the growth and development of cancer and the antimalarial effect of some of these molecules have been confirmed [START_REF] Saito | Chemical genomics identifies the unfolded protein response as a target for selective cancer cell killing during glucose deprivation[END_REF][START_REF] Anisimov | Insulin/IGF-1 signaling pathway driving aging and cancer as a target for pharmacological intervention[END_REF][START_REF] Alexandrov | The inhibition of the transplacental blastomogenic effect of nitrosomethylurea by postnatal administration of buformin to rats[END_REF][START_REF] Anisimov | Inhibition of the carcinogenic effect of 7,12-dimethylbenz(a) anthracene in female rats by buformin, phenytoin, pineal polypeptide extract and L-dopa[END_REF]. Metformin (Fig. 1a) is one of the best-known biguanide drugs used in type II diabetes. Studies on metformin have also shown its benefits for patients with specific types of cancer [START_REF] Jiralerspong | Metformin and pathologic complete responses to neoadjuvant chemotherapy in diabetic patients with breast cancer[END_REF]. Buformin and phenformin (Figs. 1b and 1c,respectively) are other anti-diabetic biguanides which also have an inhibitory effect on cancer development [START_REF] Caraci | Effects of phenformin on the proliferation of human tumor cell lines[END_REF][START_REF] Anisimov | Insulin/IGF-1 signaling pathway driving aging and cancer as a target for pharmacological intervention[END_REF][START_REF] Alexandrov | The inhibition of the transplacental blastomogenic effect of nitrosomethylurea by postnatal administration of buformin to rats[END_REF]. The delivery of these drugs to their targets is of great importance and little understood. The interaction of the biguanides referred to above and a transport protein such as β-lactoglobulin (BLG) could be a valuable factor in controlling their transport to biological sites.

Lipocalins are small proteins that have the ability to bind and transfer small hydrophobic ligands [START_REF] Flower | The lipocalin protein family: structural and sequence overview[END_REF][START_REF] Schlehuber | Lipocalins in drug discovery: From natural ligand-binding proteins to 'anticalins[END_REF]. The core structure of these proteins consists of an eightstranded anti-parallel β-barrel that defines an internal cavity for binding the ligands. BLG is a major whey protein which has several genetic variants [START_REF] Sawyer | The core lipocalin, bovine β-lactoglobulin[END_REF][START_REF] Dong | Infrared and circular dichroism spectroscopic characterization of structural differences between β-lactoglobulin A and B[END_REF]. It consists of 162 amino acids with approximately 18.4 kDa of molecular mass [START_REF] Brownlow | Bovine β-lactoglobulin at 1.8 ˚A resolutionstill an enigmatic lipocalin[END_REF]. This lipocalin protein is capable of binding hydrophobic ligands such as steroids, fatty acids, retinoids, vitamin D and cholesterol [START_REF] Kontopidis | The ligandbinding site of bovine β-lactoglobulin: Evidence for a function[END_REF][START_REF] Kontopidis | β-Lactoglobulin: Binding properties, structure, and function[END_REF][START_REF] Wu | β-Lactoglobulin binds palmitate within its central cavity[END_REF]. Identifying novel bioactive ligand molecules of BLG is of biotechnological and pharmacological importance and contributes to further understanding of the molecular-recognition properties of this lipocalin. However, BLG exhibits significant resistance against both gastric-and simulated duodenal digestions. Accordingly, it appears a prime candidate for the safe delivery and protection of pH-sensitive drugs in the stomach. The interaction and protein conformation of BLG with metformin, buformin and phenformin were investigated in the present study, using moleculardocking and molecular dynamics simulation studies. This may contribute not only to the better understanding of the molecular properties of this protein but might also influence future studies of the transport of drugs to biological sites.

Theoretical

Molecular-docking

All of the docked conformations of biguanideBLG complexes were generated using the ArgusLab 4.0.1 molecular-docking program [START_REF] Thompson | Molecular docking using ArgusLab: an efficient shape-based search algorithm and the AScore scoring function[END_REF]. A comparison of the results of ArgusLab and GOLD (Genetic Optimisation for Ligand-Docking) shows a little difference in their docking accuracies [START_REF] Thompson | Molecular docking using ArgusLab: an efficient shape-based search algorithm and the AScore scoring function[END_REF][START_REF] Nikolic | QSAR study of imidazoline antihypertensive drugs[END_REF]. The known crystal structure of BLG (PDB ID: 3NPO) was taken from the RSCB protein data bank. The ArgusLab suite was used to remove its water molecules. The Becke three-parameter Lee-Yang-Parr (B3LYP) hybrid density functional theory was employed to optimise the biguanides' structures, using the GAMESS (USA) quantum chemistry software [START_REF] Schmidt | General atomic and molecular electronic structure system[END_REF]. The uncharged form of biguanides was selected for the docking calculations. The blind-docking approach was used and the whole protein was taken as a potential binding site. The Ascore scoring method in ArgusLab, using high precision 0.4 ˚A grid resolution and a maximum of 200 candidate poses was employed for the docking calculations. The Ascore scoring function ranked the docked conformations on the basis of their free-binding energy.

The lowest free-binding energy conformation of each biguanide-BLG complex was selected for further studies.

Molecular dynamics simulation

The lowest free-binding energy conformation of each complex was selected as the initial conformation for the molecular dynamics (MD) studies. All MD studies were carried out using GROMACS4.5.6 (University of Groningen, Groningen, The Netherlands) package [START_REF] Berendsen | GROMACS: A message-passing parallel molecular dynamics implementation[END_REF][START_REF] Lindah | GROMACS 3.0: a package for molecular simulation and trajectory analysis[END_REF] and GROMOS96 43a1 force field [START_REF] Van Gunsteren | Biomolecular simulation: The GROMOS96 manual and user guide[END_REF][START_REF] Schleyer | The encyclopedia of computational chemistry[END_REF]. The Dundee PRODRG2.5 server was used to generate the topology parameters of biguanides [START_REF] Sch¨uttelkopf | PRODRG: a tool for high-throughput crystallography of protein-ligand complexes[END_REF]. The partial atomic charges of biguanides were calculated using GAMESS quantum chemistry software [START_REF] Schmidt | General atomic and molecular electronic structure system[END_REF] at the level of HF/6-31G**. The complex was located in a cubic box with the periodic boundary conditions. The box volume was 294.14 nm3 and filled with extended simple-point charge (SPC) water molecules [START_REF] Pullman | Intermolecular forces[END_REF] and the solvated systems were neutralised by adding 8 sodium ions. Energy minimisation was performed using the steepest descent method for the 4000 time-step. Next, the system was equilibrated for 40 ps at a temperature of 300 K. Finally, a 10 ns MD simulation was carried out at 0.1 MPa and 300 K. A Berendsen thermostat [START_REF] Berendsen | Molecular dynamics with coupling to an external bath[END_REF] at 300 K, the particle mesh Ewald (PME) method [START_REF] Darden | Particle mesh Ewald: An N•log(N) method for Ewald sums in large systems[END_REF][START_REF] Essmann | A smooth particle mesh Ewald method[END_REF] for long-range electrostatics, a 7 ˚A cutoff for the van der Waals interactions and Coulomb interactions were used.

The equation of motion was integrated by the leap-frog algorithm with the 2 fs time-step. The atomic coordinates were recorded to the trajectory file every 0.5 ps for subsequent analysis.

Results and discussion

Molecular-docking studies

Three potential binding sites have been reported for ligand-binding to BLG: (i) the internal cavity of the β-barrel; (ii) the surface hydrophobic pocket in a groove between the α-helix and the βbarrel and; (iii) the outer surface near Trp19-Arg124 [START_REF] Roufik | Thermodynamics of binding interactions between bovine β-lactoglobulin A and the antihypertensive peptide β-Lg f142-148[END_REF]. In this study, the binding mode of biguanides: metformin, buformin and phenformin, at the active site of BLG was examined. The docking results show that phenformin binds in the internal cavity of BLG (Fig. 2a) but metformin and buformin bind to the surface of BLG (Figs. 2b and2c). Fig. 3a shows that there is one hydrogen bond interaction between phenformin and Pro (38) with 2.97 ˚A distance. In addition, there are 4 hydrogen bond interactions between metformin and Cys(66), Glu(65) and Gln(68) with 2.97 ˚A, 2.85 ˚A, 3.02 ˚A and 2.87 ˚A, respectively. Fig. 3b shows that Gln(68) interacts with metformin by 2 hydrogen-bond interactions. One hydrogen bond interaction between buformin and Ser(30) and with 2.65 ˚A distance is shown in Fig. 3c. The near residues for each biguanide are listed in Table 1. It may be concluded that, in addition to the hydrophobic interactions which stabilise the proteindrug complexes, the hydrogen-bond interactions also play an important role in the stability of BLGbiguanide complexes. The binding constant (Ka) and freebinding energy (∆G•) of biguanides are listed in Table 2. The results showed that the interaction of BLG with phenformin was more favourable than those with metformin and buformin. This is due to the hydrophobic nature of the central cavity of BLG. Hence, it may be concluded that the hydrophobic interactions have a significant role in the binding of these drugs to BLG. The interaction of novel anti-diabetic pyrimidine-fused heterocycles and BLG was recently investigated by [START_REF] Mehraban | Binding study of novel anti diabetic pyrimidine fused heterocycles to β-lactoglobulin as a carrier protein[END_REF]. Their fluorescence spectroscopic results show that these drugs bind in the hydrophobic pocket (calyx) of the protein with a binding constant of from approximately 11 × 103 M-1 to 300 × 103 M-1. Hence, the affinity of BLG for binding the biguanides are greater than that of the pyrimidine-fused heterocycles.

MD simulation studies

The root mean square deviation (RMSD) of the backbone of BLG, the total energy of the system, the system temperature and density of the system were obtained to examine the stability of trajectories for free BLG and BLG-biguanide complexes (Fig. 4). Analysis of Fig. 4a reveals that the RMSD of various systems achieved equilibrium and fluctuated at around the mean value at various times. The equilibration time is approximately 3500 ps for free BLG, BLGphenformin and BLG-buformin, whereas the BLGmetformin complex attained equilibrium after 5000 ps. The radius of gyration (Rg) for BLG and BLGbiguanide complexes was also determined and plotted against the simulation time to examine the protein compactness (Fig. 5). The results show that the Rg of all systems stabilises after approximately 2.5 ns, indicating that the MD simulation equilibrated after 2.5 ns. The initial value of Rg for all systems is approximately 1.44 nm, which accorded with earlier experimental results [START_REF] Renard | Etude de l'agrégation et de la gélification des protéines globulaires: application `a la bétalactoglobuline[END_REF]. Fig. 5 also shows that the Rg value of BLG does not depend upon complexation with biguanides. This indicates that the environment of BLG did not change during its interaction with biguanides. Fig. 6 shows the changes in the total solvent accessible surface (SAS) of the protein during the 10 ns simulation-time. The variation in SAS reveals that the trends for BLG and BLG-biguanide complexes are similar to the variation of their Rg. This similarity validates the accuracy of the MD simulation results. In addition, the amount of secondary structure components of BLG and BLG-biguanide complexes revealed that the conformation of BLG did not change during biguanides complexation (figure is not shown).

Conclusions

On the basis of the molecular-docking results, metformin and buformin bind to the surface of BLG and phenformin binds in the internal cavity of BLG. Of these biguanides, phenformin has the most negative free-binding energy. In addition, the docking results reveal that the hydrophobic interactions play a major role in the BLG-biguanide complex stability. MD simulation studies showed the stabilisation of BLG and BLG-ligand complexes at around 2.5 ns. The BLG did not exhibit any conformational changes during the biguanides-binding. Also, the profiles of atomic fluctuations show the rigidity of the ligand-binding site during the simulation.

  The time-averaged root mean square fluctuations (RMSF) of protein residues was obtained to analyse the local mobility of the protein. The RMSF values versus the residue numbers are shown in Fig.7. These values are based on the 6.5 ns, 6.5 ns, 6.5 ns and 5.0 ns of trajectory for BLG, BLG-phenformin, BLG-buformin and BLG-metformin complexes, respectively. The fluctuations of residues in the binding sites are lower than the others. This clearly indicates that the binding site remains approximately rigid during the MD simulation.

Acknowledgements. The financial support received from the Research Council of the University of Isfahan is gratefully acknowledged.


Maria S Nikulshina

Pascal Blanchard

Alexander Mozhaev

Christine Lancelot

Anne Constant

Michel Fournier

Edmond Payen

Olivier Mentre

Valérie Briois

Pavel A Nikulshin
email: p.a.nikulshin@gmail.com

A Griboval-Constant

O Mentré

C Lamonier
email: carole.lamonier@univ-lille1.fr

Molecular approach to prepare mixed MoW alumina supported hydrotreatment catalysts using H4SiMonW12-nO40 heteropolyacids

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The Keggin-type heteropolyanion or polyoxometallate (POM) XM12O40 n-(with M = Mo, W and X a non-metallic atom) consists in a regular XO4 tetrahedron surrounded by 12 MO6 octahedra, which are connected by shared edges to form trimetallic M3O13 groups joined together by their vertices 1 . Depending on the synthesis conditions, various isomers (as α,β,γ), obtained by a π/3 rotation of a M3O13 group around C3 axis, can be isolated [START_REF] Pope | Heteropoly and Isopoly Oxometalates[END_REF] . At solid state, these ions and their counter-cations form a crystalline network, with a symmetry depending on the hydration of the solid and the nature of the counter-cation [START_REF] Pope | Heteropoly and Isopoly Oxometalates[END_REF] . The "lacunary" Keggin structure corresponds to a Keggin structure in which one or more sites previously occupied by molybdenum or tungsten atoms are vacated. This new family corresponds to an M/X ratio of 11 [START_REF] Souchay | Ions minéraux condensés[END_REF][START_REF] Keggin | [END_REF][5] or 9 6,7 , obtained by extracting one or three metal atoms from a single M3O13 group or from different M3O13 groups. These monovacant or polyvacant heteropolyanions can be synthesized directly depending on the medium pH. It is also possible to prepare substituted derivatives in which one or more metal atoms (Mo, W, etc.) of the dodeca-condensed structure are replaced by different atoms. Substitution can take place with an element whose oxidation number is much lower than that of the metal atom it is replacing (e.g. cobalt-II, nickel-II, iron-III…) [START_REF] Pope | Heteropoly and Isopoly Oxometalates[END_REF][START_REF] Souchay | Ions minéraux condensés[END_REF] , or with an element that can have similar properties to that of the substituted atom, as with vanadium-V in the case of the acid H4PMo11VO40 8 . Sanchez et al. 9 have thereby isolated potassium and tetrabutyl ammonium salts of mixed SiMo1W11O40 4-and SiMo3W9O40 4- heteropolyanion. Reduction study on mono substituted Keggin SiMo VI 1W VI 11 synthesized with microtube shape has also been recently reported by Shen and al. 10 with the formation of stable reduced SiMo V 1W VI 11 microtube. In the field of hydrotreatment catalysis, Keggin and Keggin-derivated heteropolymolybdate and heteropolytungstate structures have been successfully used to replace conventional precursors as ammonium heptamolybdate (NH4)6Mo7O24 (AHM) and ammonium metatungstate (NH4)6H2W12O40 (AMT) to prepare oxidic precursors of metal sulfide catalysts [11][12][13][14][15] . From a catalytic point of view, the use of mixed metals Keggin-type POM may enhance activity due to the introduction of a second metal in the POM cluster. Mixed HPA precursors PMo(12-x)VxO40 (3+x)-generally used in the field of mild oxidation catalysis have been prepared for their use in hydrodemetallation catalysis 16 or diesel hydrotreatment 17 . The synthesis of cobalt salt of substituted PCoMo11O40H 6-has been developed leading to HDS catalysts with improved catalytic performances compared to those of conventional catalysts prepared from AHM and cobalt nitrate 18 . For hydrocracking catalytic application, NiW ASA (Amorphous Silica Alumina) supported sulfide catalysts were also prepared from heteropolytungstate Ni salts as the substituted Ni3PW11NiO40H or the lacunary Ni4SiW11O39 salt 12 . For the CoMo system as well as for the NiW one, the vicinity of both elements in the HPA salt at the molecular level was able to generate a great amount of Co-MoS2 or Ni-WS2 active phase after sulfidation leading to improved catalytic performances 11,19 . Another way to develop active hydrotreating catalysts was carried out in 2001 with the use of bulk mixed tri-metallic (Ni)MoW sulfide. This new type of catalyst was at least three times more efficient than any previous hydrotreating catalyst, conventionally used in industry 20 . Further researches recently proposed to use less expensive MoW mixed supported catalysts prepared by impregnating on a support classical precursors (together or sequentially) such as ammonium heptamoybdate and ammonium metatungstate [START_REF] González-Cortés | Please do not adjust margins[END_REF][22][23] or ammonium thiomolybdate (NH4)2MoS4 and ammonium thiotungstate (NH4)2WS4 24 . Mechanical mixture of supported NiMo-NiW catalysts has also been proposed 25 . Whatever the preparation procedure except mechanical mixture, Ni promoted mixed MoW catalysts revealed most of the time better catalytic performances than that of their monometallic counterpart. We propose here a new approach to prepare MoW catalysts based on the use of mixed MoW heteropolyacids H4SiMonW12-nO40, allowing to start from a single molecular entity to introduce together Mo and W. We synthesized two mixed heteropolyacids H4[SiMo1W11O40] and H4[SiMo3W9O40] that were extensively characterized at the solid state combining X-Ray Diffraction and XAS (X-Ray Absorption Spectroscopy) at the Mo K and W LIII edges. Purity of these mixed HPA together with their stability in aqueous solution was checked by Raman spectroscopy and polorography. Characterizations by Raman and XAS were also performed after deposition on alumina support in order to check the preservation of the mixed heteropolyacids. Catalytic results of the mixed supported system were compared to that obtained using monometallic H4SiMo12O40 and H4SiW12O40 HPA and their mixtures with Mo/W ratio = 1/11 and 3/9. Catalysts EXAFS characterization at the Mo K and W LIII edges after sulfidation have been used to explain the different hydrodesulfurization and hydrogenation properties.

Experimental

Synthesis of mixed H4[SiMonW12-nO40] heteropolyacids (HPA) (n=1,3)

α-H4[SiMo1W11O40]

In the first step, the lacunary Keggin polyoxotungstate potassium salt α-K8[SiW11O39] was prepared following reference 26 . 182 g of sodium tungstate (Na2WO4,2H2O) and 11 g of silicate (Na2SiO3,5H2O) were dissolved in 300 mL of hot water (60°C) in which was added 195 mL of hydrochloric acid (4M). After boiling this solution during one hour and cooling at room temperature (RT), the potassium salt was precipitated by solid KCl addition (about 50 g) and separated by filtration. The synthesis of the potassium salt α-K4[SiMo1W11O40] was then performed at RT following reference 9 . A MoO2 2+ molybdenum solution was prepared by addition of 6mL of nitric acid (13M) in 10 mL of a Na2MoO4 solution (1M). Then 10 g of α-K8 [SiW11O39] were added by small fractions. The potassium salt of SiMo1W11O40 4-precipitated as a yellow solid. In order to allow better yield, it was filtered after 3 hours and purified by recrystallization in distilled water. We observed that the mixed corresponding free acid was sufficiently stable to be crystallized from aqueous solution by using the "etherate" method 2 . A concentrated aqueous solution of the mixed MoW potassium salt was strongly acidified with HCl 12M and shaken with excess of diethyl oxide at RT. After separation, the heavy etherate phase was decomposed by addition of water. The remaining solution is let one night (around 12 hours) in a fume hood allowing vapor ether extraction. After removal of ether the aqueous solution was evaporated at 4 °C until α-H4[SiMo1W11O40] heteropolyacid crystallization.

β-H4[SiMo3W9O40]

In the first step the lacunary Keggin polyoxotungstate sodium salt β-Na9[SiW9O34H] was prepared according to reference 27 . 12 g of sodium metasilicate (Na2SiO3, 5H2O) were dissolved in 250 mL of cold water (12°C) and 150 g of sodium tungstate (Na2WO4, 2H2O) were added. Hydrochloric acid (6M, 95 mL) was slowly added into the vigorously stirred solution. A silica residue was filtered off if necessary. The solution was left to stand at RT at least one hour while stirring and let in the fridge at 4°C during two days until precipitation. The synthesis of the mixed potassium salt β-K8[SiMo3W9O39] was performed following ref 9 . A MoO2 2+ molybdenum solution was prepared by addition of 60 mL of hydrochloric acid (12M) in 40 mL of a Na2MoO4 solution (1M). Then 27 g of β-Na9[SiW9O34H] were added by small fractions of 3 g. After 30 min, the resulting pale yellow solution was precipitated by 5 g of solid KCl to form K4[SiMo3W9O40] that was filtered off. The H4[SiMo3W9O40] heteropolyacid is also prepared by dissolving the mixed potassium salt in water and extraction by ether and HCl. Crystals of β-H4[SiMo3W9O40] were formed after evaporation at 4°C.

Preparation of the supported oxidic precursors

Impregnating solutions were prepared by dissolving mixed heteropolyacids in water solution. For comparison purposes, monometallic impregnating solutions were prepared using the Keggin heteropolyacids H4SiMo12O40 and H4SiW12O40 prepared according to references. [START_REF] Souchay | Ions minéraux condensés[END_REF]28 Bimetallic reference catalysts were also prepared using impregnating solutions obtained by mixing H4SiMo12O40 and H4SiMo12O40 in water solution with a Mo/W ratio of 1/11 and 3/9 corresponding to the mixed MoW heteropolyacids and were denoted Ref Mo1W11 and Ref Mo3W9, respectively. The oxidic precursors were prepared by incipient wetness impregnation of alumina support [γ-Al2O3 (Norton), specific area: 240 m 2 /g, pore volume: 0.9 mL/g] with the aforementioned impregnating solutions.

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In any case for comparison purpose, the HPA concentration was kept at 0.145 molHPA.L -1 to set the (Mo+W) surface density at 3.9 atoms /nm 2 , leading to different theoretical Mo/W loadings as reported in Table 1. All prepared impregnation solutions had a close pH value around 0.25 due to the same concentration of protons. After impregnation and 2 h of maturation in a wet atmosphere in order to let the species diffuse into the alumina extrudates, the solids were dried overnight at 100°C without further calcination.

Raman Spectroscopy

The Raman spectra of the samples were recorded at RT using an Infinity XY Horiba Jobin-Yvon Raman microprobe equipped with a photodiode array detector. The exciting laser source was the 532.16 nm line of a Nd-YAG laser. The wavenumber accuracy was 4 cm -1 .

TGA and DSC analysis

Thermogravimetric analysis was carried out with a STA 449 F3 Jupiter. The measurements were conducted in air with heating from RT to 600°C and a heating rate of 10°C/min. Samples of about 30 mg were introduced into a small corundum crucible, an empty crucible was used as the standard for thermal analysis.

Single crystal X-Ray Diffraction

Single crystals suitable for XRD experiments have been isolated from the crystallization products and sticked on a glass fiber using vacuum grease. Crystals of β-H4[SiMo3W9O40].9H2O are air stable and can be handled without precaution. On the opposite, crystals of the α-H4[SiMo1W11O40].30H2O are very unstable under exposure in air and rapidly decompose. In this case, crystals were placed in grease, rapidly mounted and the data were collected under nitrogen flow at 100 K without any decomposition. Both crystal structures have been refined using single crystal XRD data collected on a DUO-Bruker SMART apex diffractometer using the Mo Kα radiation by least square minimization on Fhkl. Intensity data are corrected for Lorenz-Polarization effects and absorption, then the structure was solved and refined. Multiscan absorption correction was applied using SADABS [START_REF] Sheldrix | SADABS: Area Detector Absorption Correction[END_REF] . The structure was solved using Superflip [START_REF] Palatinus | [END_REF] and refined using Jana 2006 31 . Specificities for each collection and refinement are given below in the crystal structure dedicated sections. Further details of the crystal structures may be obtained from the Fachinformationszentrum Karlsruhe, 76344 Eggenstein-Leopoldshafen, Germany (Fax: +49-7247-808-666; e-mail: crysdata@fiz-karlsruhe.de, http://www.fizkarlsruhe.de/request_for_deposited_data.html) on quoting the depository numbers CSD-431628 and CSD-431629 for the SiMo1W11O40H4 and SiMo3W9O40H4 hydrates, respectively. The quantification of the water molecules is discussed in the dedicated section.

X-Ray Absorption Spectroscopy

Mo K and W LIII edges EXAFS (Extended X-ray Absorption Fine Structure) measurements were carried out at the ROCK beamline of the SOLEIL synchrotron source 32 using Si(111) monochromator. The measurements were done at RT on powdery sample pressed into pellets when characterizing bulk HPA and dried catalysts at the oxide state. Sulfide catalysts were characterized after in-situ sulfidation using the catalytic cell available at SOLEIL 33 . Powdered dried oxide catalysts were loaded in the cell and the in-situ sulfidation was performed with a flow of 10% H2S in H2 at atmospheric pressure from RT to 400°C with a plateau of two hours according to the activation procedure applied in catalytic tests. For the whole measurements, XAS spectra were obtained in transmission mode with ionization chambers as X-Ray detectors. The ionization chambers were filled with a mixture of argon and nitrogen (50:50) for measurements at the Mo K edge and (73:27) at the W LIII edge. Mo and W edges EXAFS regions of the spectra were extracted and analyzed using Athena and Artemis software packages 34 . For Mo K edge, k 3 χ(k) EXAFS signals were Fourier transformed in R-space pseudo-radial distribution functions using a Kaiser-Bessel window between kmin = 4.8 Å -1 and kmax = 10.5 Å -1 and dk window sill parameter corresponding to the sloped region of Fourier transform is equal to 2. For W LIII edge, FT of the k 3 χ(k) EXAFS data were carried out from 3.5 to 16.8 Å -1 with a dk parameter equal to 2. The inverse Fourier Transformation of the EXAFS at the Mo K edge and W LIII edge were fitted using appropriate single and multiple scattering EXAFS This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins paths generated from the cif files derived from crystallographic structures. During fitting of mixed bulk samples using Artemis, the distances between the concerned atoms and the coordination numbers were constrained by the related parameters extracted from the XRD single crystal data to check the transferability of amplitude and phase functions which will be further applied for catalysts EXAFS analysis. In this frame, the amplitude reduction factor S0 2 , which takes into account multielectronic effects, together with the enot parameter which allows to match the k-scale of theoretical phase and amplitude functions with the one of the experimental EXAFS signals related to the user choice of the E0 position on the edge, were first determined on the bulk HPA for the Mo and W edges. S0 2 and enot values were after kept fixed for catalysts fitting. The number of fitted points was below the number of independent points according to the Nyquist criterion 35 . Typical accuracies for the determination of the coordination numbers and distances are 10 to 20 % and 1 to 2 %, respectively.

Polarography

The polarographic measurements were performed on a Tacussel (Pol 150) electrochemical analyzer monitored by a Tracemaster 5 programmer. A conventional single-compartment cell with standard three-electrodes configuration was used. Pt was used as an auxiliary electrode, while Ag/AgCl was used as a reference electrode. The working electrode was a glassy carbon disk (using a rate of stirring was equal to 840 rpm) allowing the heteropolymolybdates study with reduction waves observed in the range -0.1 and 0.5 V. Tungsten compounds being less reducible than molybdenum compounds, the study of H4SiW12O40 that requires a mercury dropping electrode creating a higher overvoltage for reaching lower potential (between -1.1 and -0.2 V) could not be performed. Deoxygenating of the solutions was carried out using argon as inert gas. The concentration in molybdenum atoms was equal 10 -3 M in aqueous electrolytic solutions HCl (1 M)/ethanol (equivolume).

XPS analysis

The composition and chemical state of the elements were studied using X-ray Photoelectron Spectroscopy (XPS) on a KRATOS Axis Ultra spectrometer equipped with a monochromatic Al Kα (1486.6 eV) Xray radiation (powered at 100 watts). The analysis chamber was operated under ultrahigh vacuum with a pressure close 10 -10 Torr. The binding energy (BE) values were referred to the Al2p at 74.6 eV. Simulation of the experimental photopeaks was carried out using a mixed Gaussian/Lorentzian peak fit procedure according to the software supplied by Casa XPS. The methodology implemented for the decomposition of the Mo 3d and W4f spectra of the sulfided solids has been described elsewhere 12,36 . The Mo3d spectrum is decomposed in three doublets regarding Mo (three Mo species) and two S2s components associated to two sulfur species S 2-and (S2) 2-as usually reported for Mo(W) and Co(Ni)Mo hydrotreatment catalysts. The three well known Mo doublets whose Mo3d5/2 components are placed at about 229.0, 230.5 and 233.0 eV according to three oxidation states of molybdenum, Mo IV , Mo V and Mo VI respectively. The first contribution is attributed to MoS2 phase, the second one is attributed to Mo V surrounded by oxygen and sulfur atoms (intermediate sulfidation phase) and the contribution at higher binding energy is assigned to Mo VI oxide species. The W4f spectra could also be decomposed into three doublets whose W4f7/2 components are attributed to three different W species: W IV sulfide (WS2) with BE at about 32.2 eV), W V oxysulfide with BE at about 33.5 eV and W VI oxide with BE at about 36 eV. The decomposition of Mo3d and W4f XPS spectra allowed obtaining the relative atomic % of each corresponding Mo (W) species noted %Moi (%Wi) according to the following formula:

%Moi = [Ai(Mo3d5/2 + Mo3d3/2) /∑(Ai(Mo3d5/2 + Mo3d3/2))] × 100
where Ai(Mo3d5/2 + Mo3d3/2) is the area of one Mo doublet, and %Wi = [Ai(W4f7/2 + W4f5/2) /∑(Ai(W4f7/2 + W4f5/2))] × 100 where Ai(W4f7/2 + W4f5/2) is the area of one W doublet.

Catalytic activity

The catalytic properties were evaluated using a fixed-bed microreactor featuring a high-pressure flow system. 0.6 g of sieved catalyst (0.25 -0.50 mm) was diluted with 1 cm 3 of low-surface-area sieved carborundum (0.2 -0.4 mm) and placed in the centre of the reactor (the reactor had an internal diameter of 8 mm). Before testing, the catalysts were sulfided in a flow of 10% H2S in H2 at 400°C for 2 h. A mixture of DBT (1500 ppm S), naphthalene (3 wt. %), hexadecane (as an internal standard, 1 wt. %) and toluene (as a solvent) was used as a model feedstock for evaluation of HDS and hydrogenation (Hyd) performances. Catalysts were tested under the following conditions: 320°C, 3.0 MPa hydrogen, 10 h -1 liquid hourly space velocity (LHSV) and a 500 NL/L volume ratio of hydrogen to feed. The liquid product compositions of the samples collected every 0.5 h were determined using a Crystall-5000 Gas Chromatograph (GC) equipped with a 30 m OV-101 column. The reaction products were identified by matching retention times with those of commercially available standards and by GC/MS analysis using a Finnigan Trace DSQ. The HDS reaction was allowed to proceed for 12 h to evaluate the deactivation of the catalyst. However, all catalysts exhibited stable performance, achieving a steady state after 5 -6 h. All catalysts were tested two times to exclude the possible error and average values of reactants conversion and selectivity ratios were calculated. The rate constants of the pseudo-first-order reactions of the DBT HDS and naphthalene Hyd were determined using the following equations:

) 1 ln( DBT DBT HDS x w F k    ) 1 ln( Naph Naph x w F k Hyd    , (1)
where KHDS and KHyd are the pseudo-first-order reaction constants for the DBT HDS and naphthalene Hyd (mol g -1 h -1 ) respectively, XDBT and xNaph are the conversions (%) of DBT, and naphthalene respectively, F DBT and FNaph are the reactant flows in moles (mol h -1 ) and w is the weight of the catalyst (g). The HDS products from DBT include biphenyl (BP) via the direct desulfurization (DDS) pathway, as well as cyclohexylbenzene (CHB)

Please do not adjust margins Please do not adjust margins and bicyclohexyl (BCH) from the HYD pathway. The HYD/DDS selectivity was calculated according to the reaction network for DBT HDS (Scheme 1):

BP BCH CHB DDS HYD / C C C k k S DDS HYD    ,
(2) where CCHB, CBCH, and CBP are the concentrations (wt. %) of CHB, BCH, and BP in the reaction products, respectively. Scheme 1. Reaction network of the HDS of DBT.

Results and discussion

HPA Crystal Structures:

For both compounds, the space group was determined by means of the systematic extinctions and several models have been tested in case of ambiguities. The charge flipping method located most of the atoms with their chemical nature on the basis of their coordination and convergence of the refinement. Complementary anions, including water molecules have been located on Fourier-difference maps. They are often associated to large thermal parameters due to local disorder, e.g. Mo/W, protonation or not. To validate the presence of water molecules in between the polyanions, the occupancies of oxygen atoms (Ow) was refined and rejected if significantly lower than 100%. It means that the hydration rate given above stem from crystal refinements, but additional water molecules could exist in some partially occupied available voids. Collection and refinement parameters are given in Table 2 37 and the α-PMo9O34H6 3- structure determined by Strandberg 38 , showing in both cases three adjacent metallic sites. Based on these literature data, Sanchez et al. 9 assumed that in the β SiMo3W9O40 4-the three Mo atoms belonged to three different groups without performing any structural determination.

Please do not adjust margins

Please do not adjust margins β-H4[SiW9Mo3O40] EXAFS oscillations both at the Mo K and at the W LIII edges have been successfully refined (see supporting information) taking into account the radial distribution functions of neighbors deduced from the crystallographic data determined from XRD single crystal analysis. In Tables 3 and4 are reported the main parameters of the least square fitting process at the Mo K and W LIII edges, the number of neighbors N, the distance R (Mo-X for Table 3 and W-X for Table 4), the Debye-Waller factor σ² and the R factor which measures the relative misfit of the simulation with the experimental data. In agreement with N and R crystallographic data also reported in Table 3 the Around W (Table 4), the first coordination shell of bulk H4[SiW9Mo3O40] presents similar oxygen contributions than that observed around Mo due to similar size of six-fold coordinated Mo 6+ (0.59 Å) and W 6+ (0.60 Å) cations 39 . The second coordination shell around W contains five contributions, the second neighbors around W are shared between W (N=1.33) and Mo (N=0.66) at 3.36 and 3.40 Å from the same M3O13 group while W neighbors belonging to another M3O13 group are shared between W from the 60° rotated W3O13 group and the three others groups leading to two different distances equal to 3.74 Å and 3.68 Å respectively.

H4[SiMo1W11O40]

α-H4[SiMo1W11O40] EXAFS oscillations at the Mo K edge have also been successfully refined (see supporting information (SI)) using the radial distribution function extracted from crystallographic data deduced from XRD single analysis. In Table 3 the disordered octahedral coordination of Mo is described with two contributions of three oxygen neighbors at 1.79 and 2.00 Å. At the W LIII edge, due to the α symmetric structure, in addition to the Si contribution, only a unique W contribution at 3.47 Å is present in the second coordination shell. It was not possible to refine EXAFS oscillations at the W LIII edge for distances above the first coordination shell by keeping R and N crystallographic values. In this symmetric α structure, the part of multiple scattering is so important that a high contribution of paths involving at least 3 atoms as W-O-W has to be taken into account making the parameters to fit higher than the number of independent points in the fit. These EXAFS results confirmed the formation of the mixed HPA.

IR, Raman and TGA-DSC analysis (SI)

IR and Raman spectra of H4[SiMo1W11O40], H4[SiMo3W9O40], H4[SiMo12O40] and H4[SiW12O40] are reported in SI. The IR spectra as well as the Raman ones of mixed HPA are very close and look like that of H4SiW12O40 indicating that in the mixed HPA Mo-and W-oxygen vibrational modes cannot be observed separately. TGA-DSC analysis (see SI) shows that the thermal behavior of the mixed HPA and silicotungstic acid is very similar and that the substitution of W by Mo slightly decreases the thermal stability of the mixed HPA compared to that of silicotungstic acid.

HPA impregnating solutions analysis

Synthesis of mixed MoW HPA was devoted to the preparation of aqueous impregnating solutions for the preparation of MoW hydrotreatment catalysts possessing a MoW nanoscale proximity. It was then a key point to check the preservation of mixed heteropolyacids after their dissolution in water.

Backscatterer N R (Å) σ² x 10 Please do not adjust margins Please do not adjust margins

Raman spectroscopy

Raman spectra of impregnating solutions (Fig. 2) are in good agreement with those of the corresponding bulk compounds reported in SI. The similarity between the Raman spectra of the solid and of the solution of mixed HPA indicates i) the purity of the mixed solids with no formation of separate monometallic heteropolyanion and ii) their preservation after dissolution in water. As for Raman characterization at the solid state, the wavenumber of M=Ot vibration (main peak of the Raman spectrum) corresponds to a weighted average of the Mo=Ot and W=Ot wavenumbers.

Polarography

The electrochemistry of heteropolycompounds in solution has been studied for a long time by polarography. [START_REF] Pope | Heteropoly and Isopoly Oxometalates[END_REF][START_REF] Souchay | Ions minéraux condensés[END_REF][START_REF] Souchay | Polyanions et Polycations[END_REF] It consists in observing the reduction waves (current (I)-potential (E) curves) corresponding to successive electron exchanges. In this work, due to the use of the glassy carbon disk as working electrode, the polarogram of H4SiW12O40 solution where reduction waves appear between -0.3 and -1.0 V 28 cannot be observed. Fig. 3 shows the polarograms of the three HPA solutions obtained by dissolving bulk H4SiMo12O40, H4SiMo1W11O40 and H4SiMo3W9O40 in a buffer medium. In the Keggin-like structure the electrons are delocalised over the entire polyanion, and Mo-O-Mo bridges allow the paired electrons to move according to the bipolaron model 9 .The degrees of reduction of the heteropolymolybdates are referred to as 0, II, IV, VI… in which the roman number indicates that the anion is reduced by 0, 2, 4, 6 or more. In acidic medium, three "2-electron" reduction waves (O-II, II-IV, IV-VI) can be seen in the polarogram of H4SiMo12O40 under our experimental conditions (AgCl/Ag reference electrode and HCl 1 mol.L -1 /ethanol 50-50 vol electrolyte solution) with half-wave potentials recorded at 0.280, 0.190 and -0.020 V respectively. The global shape of the polarogram and the half-wave potentials values together with their differences are in agreement with literature data 1-3,28 and confirm the presence of α-H4SiMo12O40 isomer. Polarography is one of the techniques allowing us to differentiate between α and β isomers (the first half-wave potential is expected around 0.380 V for the β-H4SiMo12O40 isomer). For the two mixed HPA solutions, polarograms are clearly different from that of α-H4SiMo12O40 and only one "2-electron" reduction wave (corresponding to the O-II) reduction is visible without any contribution of H4SiMo12O40 anion. It means that all the waves are significantly shifted toward lower potential due to the presence of W in mixed HPA and the two other waves are shifted before -0.2 V and thus do not appear on the Figure 3. Taking into account that H4SiMo3W9O40 mixed HPA is a β isomer the presence of W greatly influences the Mo redox potential allowing to distinguish unambiguously between both mixed HPA. Moreover, the two welldefined polarograms of mixed samples indicate the presence of one type of HPA in solution in agreement with the existence of pure MoW mixed HPA.

Characterization of supported oxidic precursors

Raman spectroscopy

To understand the behaviour of these HPA after deposition on the support, Raman spectra of catalysts after maturation and drying with that of the corresponding bulk HPA are reported on Fig. 4. Different behaviours depending on the catalyst can be observed. For SiMo12/Al the spectrum of the solid after maturation does not correspond to that of the bulk SiMo12O40 4-anion and new Raman lines are clearly observed at 948, 564, 350 and 215 cm -1 . These lines are unambiguously assigned to the formation of the well-known AlMo6O24H6 3-Anderson heteropolyanion species is formed through decomposition of SiMo12 Keggin structure on the support due to the "buffer effect" of alumina and Al 3+ extraction from the support [START_REF] Le Bihan | [END_REF]42 . This species is maintained after drying and welldispersed on the alumina surface according to the broad Raman lines of the spectrum 11 . During impregnation the important parameter is the pH of the solution in the pore of alumina which evolves due to the PZC of alumina (around 7). respective stability of tungsten and molybdenum based heteropolyacids in aqueous solutions at various pH have been studied in the literature demonstrating that tungsten based HPA are more stable than the molybdenum ones upon pH increase [START_REF] Souchay | Ions minéraux condensés[END_REF]43 . Indeed, tungsten containing catalysts show different behaviour since the spectra of the oxidic precursors are very similar to those of their bulk precursor. For the three W based catalysts, after maturation and drying only a shift (≈20 cm -1 ) of the main line (M=Ot vibration) towards lower wavenumbers is observed (from 1000 to 982 cm -1 for SiW12/Al; from 998 to 979 cm -1 for SiMo1W11/Al and from 997 to 977 cm -1 for SiMo3W9/Al). Such shifts have already been observed after drying but in a lesser extent in the case of ASA supported heteropolytungstate and were attributed to interaction with the support 12 . Moreover, analysis of the low Raman shift region (below 260 cm -1 ) shows similar lines in shape and position for the bulk and the corresponding maturated and dried W based HPA catalysts. The line around 220-230 cm -1 attributed to the stretching mode of the W(Mo)-O vibration (where O links W(Mo) and Si) and is characteristic of Keggin structure 28 . The global Raman analysis of catalysts after maturation and drying suggests the of the mixed MoW heteropolyanion species on alumina support even if the broadness of the Raman peaks could prevent a clear discrimination between SiW11 lacunary structure and SiW11Mo Keggin structure.

EXAFS analysis

In order to confirm the preservation of the mixed HPA on alumina upon impregnation and drying, EXAFS experiments of dried catalyst have been performed at the W LIII and Mo K edges. For easier comparison purposes 5 reports EXAFS oscillations as well as Fourier Transform moduli at both edges for the dried catalysts and their corresponding bulk HPA. At the W LIII edge, Fig. 5 (a) and(b) show clearly that EXAFS signals and the corresponding Fourier transforms moduli of the dried samples SiMo1W11/Al and SiMo3W9/Al are both in phase with that of their corresponding mixed HPA. A successful fitting of SiMo3W9/Al EXAFS data with crystallographic data of its bulk starting material has been then obtained and reported in Table 4 attesting that the local order W is similar in the supported catalysts to the one of the bulk phase. This is a strong evidence of the preservation of this HPA on alumina after drying. As aforementioned, for H4SiMo1W11 due to important multiple scattering contributions for this symmetric structure it was not possible to fit the EXAFS data at the W LIII edge. Nevertheless, from Fig. 5 (a) and (b) it is clear that the bulk starting HPA matches very well with its catalyst, indicating unambiguously it is maintained on alumina surface. Regarding the Mo side (Fig. 5 (c) and (d)) if we compare dried with the bulk starting materials, similarities are observed in both EXAFS oscillations and FT even if the fitting results reported in Table 3 show some variations in the distances between Mo and its neighbours compared to that observed in the bulk materials. These results obtained from the Mo side could be explained by a partial decomposition of SiMo3W9 HPA with the removal of one Mo atom from the Keggin structure. Please do not adjust margins Please do not adjust margins

Characterization of supported sulfide catalysts EXAFS analysis

All prepared Mo(W)/Al catalysts were in-situ sulfided during synchrotron measurement sessions, with the same conditions than those used for activation of the solids prior to catalytic evaluation. The Fourier transform moduli data recorded for the samples at the end of the sulfidation are presented in Figure 6 and the corresponding fit EXAFS parameters are gathered in Table 5 and 7. At the Mo and W edges of all samples, the first contribution is located around 1.9 Å (phase-shift not corrected) on the FTs of the EXAFS signals and corresponds to S neighbors with a Mo-S or W-S distance equal to 2.41 Å, identical to that reported for bulk sulfided MoS2 or WS2 samples 44,22 . The second coordination shell, attributed to a metallic neighbor (Mo or W), is characterized by a distance around 3.17 Å, again identical to that of the bulk sulfide material 42,44 . Nevertheless, this second contribution a significant change in magnitude is observed at both edges especially for the mixed catalyst compared to the monometallic ones. Slabs size of the MoS2 or WS2 phases could influence the magnitude of the second coordination shell, a decrease of the slabs size could be then by the decrease NMo-Mo or NW-W coordination number. Results of TEM characterization after liquid phase sulfidation showed This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins that SiMo12/Al, SiW12/Al and SiMo3W9/Al catalysts presented similar average slabs sizes with values of 4.7, 4.9 and 4.8 Å respectively 45 . It could be expected that gas sulfidation could also lead to similar average slabs length for these three catalysts. Nevertheless, the effect of dispersion (slab size) on the coordination number, the local ordering of the MoS2 phase may also influence the coordination number and the introduction of some disorder (through the Debye-Waller factor) in the position of Mo atoms in a MoS2 slab can explain low Mo-Mo coordination numbers 46 . Moreover, the larger decrease observed for mixed HPA based catalysts could be due to the presence of W neighbors for Mo and vice-versa, that is due to the destructive interference of the individual scattering paths including either the absorbing atom and Mo or the absorbing atom and W which are out of phase 44,22 .

It is thus necessary to fit the data to obtain reliable quantitative local coordination of Mo and W atoms (Table 5 and6). For the MoxW12-x solids, a Mo-W contribution had to be considered for an optimum fitting of the EXAFS spectra, with 2.9 and 1.5 W neighbors at the Mo edge and 0.7 and 1.5 Mo neighbors at the W edge, for SiMo1W11/Al and SiMo3W9/Al respectively. Contrariwise, introducing a Mo-W does not improve the fitting in mixed MoW reference catalysts. Sulfide catalysts prepared from mixed heteropolyanions present Mo and W together closely coordinated which clearly demonstrates the quantitative formation of mixed Mo-W sulfide slabs. The formation of such mixed slabs may be favored by the nanoscale proximity of both metals in the heteropolyanionic structure. Recent literature discussed about mixed MoW disulfide phase formation when using different preparation and/or sulfidation procedures. Starting from separate Mo and W precursors to prepare bulk Ni promoted and unpromoted catalysts and using as sulfidation procedure 10 vol.% H2S into H2 under at 18 bar, Hein et al. 47,48 demonstrated by XAS the formation of mixed slabs from EXAFS refinements at the Mo K and W LIII edges. Haandel et al. 22 also deduced through EXAFS analysis mixed slabs formation for Ni-Mo-W catalysts prepared from conventional precursors and activated under gas sulfidation. This mixed phase has been recently clearly visualized for the first time thanks to HAADF images revealing through Z contrast W and Mo atoms in the same sulfide slabs 45 . This study was performed on SiMo3W9/Al and its corresponding reference catalysts activated by liquid phase sulfidation and showed mixed MoWS2 phase in both cases with the presence of monometallic slabs for the reference catalyst. In our case using gas sulfidation we can deduce predominant mixed sulfide phase formation using mixed HPA while monometallic WS2 and MoS2 phases are the main ones using separate monometallic HPA. Please do not adjust margins Please do not adjust margins performance of Ni promoted mixed MoW with low Mo content (Mo/W≤1) were comparable to that of the catalysts containing only W, as we observed for our Ref Mo1W11 44 . Our catalytic results allow us to show that the origin of the catalytic improvement observed for MoxW12-x is not only due to the simultaneous presence of Mo and W as in Ref catalysts but also arises from close interaction of both metals in the mixed HPA precursor leading to the formation of mixed MoxW1-xS2 active phase, as supported by XAS analysis.

Mo K edge W LIII edge

Conclusions

In this study, mixed MoW heropolyacids α-H4[SiMo1W11O40] and β-H4[SiMo3W9O40] have been synthesized from their corresponding mixed potassium salts starting from mono and tri-vacant heteropolytungstates. Crystallographic structures have been obtained and the corresponding data were used to fit with success EXAFS oscillations at the Mo K edge showing typical Mo-W distances for both mixed heteropolyacids. purity of the samples was checked by characterizations at the liquid state using Raman spectroscopy and polarography. These mixed HPA were used for preparing hydrotreatment catalysts. After deposition on alumina, mixed HPA seems to be preserved even if a partial decomposition to lacunary Keggin species may occurs and their use ensure the nanoscale proximity between Mo and W. This close vicinity between Mo and W explains the formation after sulfidation of mixed MoW sulfide slabs for mixed HPA based catalysts as demonstrated by EXAFS fitting at the Mo K and W LIII edges. Catalytic performances in dibenzothiophene HDS and naphthalene hydrogenation have been evaluated. Catalytic results confirm the beneficial use of mixed HPA and particularly β-H4[SiMo3W9O40] compared to the use of a mixture of monometallic Mo and W HPA precursors. Higher hydrogenation properties are explained by the mixed (MoW)S2 active phase formation.

Figure 1 .

 1 Figure 1. Crystals and detailed heteropolyanion structures and formation of α-H4[SiMo1W11O40] (a,b,c) and β-H4[SiMo3W9O40] (d,e,f,g). Bulk HPA analysis EXAFS analysis of bulk HPA H4[SiMo3W9O40]

  first shell around the Mo atom contains three contributions due to three Mo-O different distances (1.68, 1.91, 2.38 Å) resulting from a distorted oxygen octahedral coordination whereas the second shell contains the contribution of W, Si and Mo backscattering neighbors at 3.40, 3.58 and 3.81 Å respectively. The second neighbors at 3.40 Å are W atoms from the same M3O13 group while Mo neighbors belonging to another M3O13 appeared at higher distance (3.81 Å).

Table 4 .

 4 Structural parameters-determined by least square fitting of the inverse Fourier Transform (R = 1-4 Å) of the W LIII edge EXAFS signals for bulk H4[SiW9Mo3O40] HPA and its corresponding dried supported catalyst with S0 2 =0.76 and enot = 8.6 eV for a E0 value used for EXAFS extraction equal to 10206.70 eV. Values given in bracket correspond to the values range determined from single crystal XRD data. Italic parameters were kept fixed during the minimization process.

Figure 2 .

 2 Figure 2. Raman spectra of HPA based impregnating solution.

Figure 3 .

 3 Figure 3. Polarograms of Mo containing HPA in 0.5 M ethanol/HCl mixture.

Figure 4 .

 4 Figure 4. Raman characterisation of catalysts after maturation (Mat.) and drying (Dried) and their corresponding bulk HPA (Bulk).

Figure 5 .

 5 Figure 5. EXAFS oscillations at (a) W LIII edge, (c) Mo K edge and the corresponding Fourier Transform modulus at (b) W LIII edge (d) Mo K edge for dried mixed MoW catalyst in red and its bulk mixed HPA in blue.

Figure 6 .

 6 Figure 6. EXAFS at the Mo K edge and W LIII edge and the corresponding Fourier transforms of sulfided Mo(W)/Al catalysts.

Table 5 :

 5 Parameters obtained from the fit of the Mo K edge EXAFS spectra of the sulfided Mo(W)/Al catalysts at the Mo K edge (Δk=3.5-15 Å -1 , R-range 1.5-3.35 Å, S 2 0 = 1.05 and enot = 1.6 ± 2 eV for a E0 value used for EXAFS extraction equal 20004.3 eV).

Figure 7 .

 7 Figure 7. XPS Mo 3d and W 4f spectra recorded for Mo(W)/Al catalysts; in blue: Mo(W) 6+ oxide contributions; in pink: Mo(W)SxOy contributions; in green: Mo(W)S2 contributions (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article).

  Binding energy (eV) This journal is © The Royal Society of Chemistry 20xx

Table 1 .

 1 Composition of precursors and prepared catalysts.

	Precursor(s)	Catalyst nomenclature	MoO3 (wt %)	WO3 (wt %)
	H4SiMo12O40	SiMo12/Al	18.0	-
	H4SiW12O40	SiW12/Al	-	26.2
	H4SiMo1W11O40	SiMo1W11/Al	1.4	24.2
	H4SiMo3W9O40	SiMo3W9/Al	4.2	20.1
	H4SiMo12O40+H4SiW12O40	Ref Mo1W11	1.4	24.2
	H4SiMo12O40+H4SiW12 O40	Ref Mo3W9	4.2	20.1
	Characterization techniques

Table 2 .

 2 . Intra HPA cation-anion bond distances are reported in SI (Table 1 SI). Crystal data, data collection and structure refinement parameters for single crystal of α-H4[SiMo1W11O40] and β-H4[SiMo3W9O40] poly-hydrates. After data collection at 100K, the crystal structure was satisfactorily refined in the tetragonal P-4n2 symmetry. The lattice parameters and final R values are a= 12.735(1) Å, c=18.079(1) Å, Z= 2, Robs =2.29%, wRall =2.75%. The refinement of the statistic disorder over the three 8(i) general positions occupied by metal ions matches well the experimental predicted formula and was fixed to ideal Mo:W =1/12 : 11/12 in the last refinement cycles. Some external oxygen (O1, O5, O6) and water molecules have very large thermal parameters which mainly picture local disordering due to the statistic Mo/W disorder and distribution of bond lengths. Si-O distances are 1.624 Å while the three independent W/MoO6 octahedra are typical

	Formula	α-H4[SiMo1W11O40]•30H2O	β-H4[SiMo3W9O40].9H2O
	Diffractometer: Bruker ApexII, Duo,  =0.7107 Å (Mo Kα)
	Crystal System	Tetragonal	Monoclinic
	Space group	P-4n2	P21/m
	a (Å)	12.735(1)	10.1951(2)
	b (Å)	-	14.4697(2)
	c (Å)	18.079(1)	14.8556(2)
	β(°)	-	100.3961(8)
	V (Å 3 )	2932.2(4)	2155.52(6)
	Z	2	2
	ρcalc (g cm -3 )	3.8133	4.28
	μ (mm -1 )	21.85	24.89
	N collected Reflection	126997	29958
	N ind Reflections (all)	3013	5729
	Rint (%)	5.01	4.14

=4.10%. In the solid, the polyanions are separated by ~9.6 Å leading to a much smaller hydration ratio than in the previous case. In this compound one rare characteristic concerns the full ordering detected between Mo and W sites. The β-SiMo3W9O40 is obtained from a β-SiW9O34H lacunary structure further completed by 3 Mo atoms. The β-SiW12O40 structure differs from the Keggin α one by rotating one W3O13 group by 60° under a ternary axis and in this case each WO6 of this rotated group is sharing oxygen corners with WO6 octahedra belonging to the same W13O13 group. As shown in the mixed HPA structure presenting three adjacent Mo octahedra, the initial β-[SiW9O34H] structure was obtained by removing three adjacent W atoms from three different W3O13 groups (that differ from the rotated W3O13) in agreement with the β-[SiW9O34H] 9- structure reported by Robert and Tézé

Table 3 .

 3 Structural parameters determined by least square fitting of the inverse Fourier Transform (R = 1-3.9 Å) of the Mo K edge EXAFS signals for bulk H4[SiW9Mo3O40] and H4[SiW11Mo1O40] HPAs and their corresponding dried supported catalysts with S0 2 =1.0 and enot = 8.70 eV for a E0 value used for EXAFS extraction equal to 20012.6 eV. Values given in bracket correspond to the values range determined from single crystal XRD data. Italic parameters were kept fixed during the minimization process.

	Backscatterer	N	R (Å)	σ² x 10 3 (Ų)	R-factor
			Bulk H4[SiMo3W9O40]
	O	1.0 (1)	1.68 (1.67)	1.7
	O	4.0 (4)	1.91 (1.87-2.00)	10.1
	O W	1.0 (1) 2.0 (2)	2.38 (2.37-2.39) 3.40 (3.35-3.37)	0.5 6.3	0.0076
	Si	1.0 (1)	3.58 (3.55-3.57)	2.0
	Mo	2.0 (2)	3.81 (3.72-3.78)	4.3
			SiMo3W9/Al
	O	3.0	1.75	7.0
	O	2.0	2.02	1.0
	O W	1.0 2.0	2.37 3.42	1.5 10.6	0.0027
	Si	1.0	3.60	5.9
	Mo	2.0	3.82	13.3
			Bulk H4[SiMo1W11O40]
	O	3.0 (3)	1.79 (1.66-1.85)	17.2
	O W	3.0 (3) 4.0 (4)	2.00 (1.95-2.40) 3.47 (3.51-3.53)	2.7 5.1	0.0259
	Si	1.0 (1)	3.49 (3.52-3.53)	3.5
			SiMo1W11/Al
	O	3.3	1.74	4.0
	O W	2.7 4.0	1.96 3.40	3.9 5.5	0.0670
	Si	1.0	3.32	5.5

  This

				3 (Ų)	R-factor
			Bulk H4[SiMo3W9O40]
	O	1.0 (1)	1.69 (1.69-1.73)	1.1
	O	4.0 (4)	1.90 (1.88-1.94)	5.0
	O	1.0 (1)	2.29 (2.30-2.36)	2.5
	W Mo	1.33 (1.33) 0.66 (0.66)	3.36 (3.30-3.36) 3.40 (3.35-3.37)	6.3 7.8	0.1290
	Si	1.0 (1)	3.51 (3.50-3.55)	0.2
	W	1.33 (1.33)	3.68 (3.64-3.66)	4.4
	W	0.66 (0.66)	3.74 (3.71-3.74)	2.1
			SiMo3W9/Al
	O	1.0	1.72	0.7
	O	4.0	1.89	5.5
	O	1.0	2.32	4.7
	W Mo	1.33 0.66	3.36 3.48	6.6 5.2	0.1111
	Si	1.0	3.51	5.3
	W	1.33	3.65	4.1
	W	0.66	3.77	3.2

journal is © The Royal Society of Chemistry 20xx

Table 6 :

 6 Parameters obtained from the fit of the W LIII edge EXAFS spectra of the sulfided Mo(W)/Al catalysts at the W LIII edge (Δk=4.3-16.3 Å -1 , R-range 1.4-3.4 Å, S 2 0 = 0.87 and enot = 8.90 ± 2.0 eV for a E0 value used for EXAFS extraction equal to 10218.8 eV).

	XPS	varying from 4.2 to 18 MoO3 wt % has no influence on the sulfidation
		rate. The Ref Mo3W9 catalyst possesses the highest MoS2 sulfidation
	Figure 7 shows the decomposition of the Mo3d and the W4f spectra	rate reaching 91 %. A low sulfidation degree of tungsten (49 %) is
	for sulfided SiMo12/Al, SiW12/Al, SiMo3W9/Al and Ref Mo3W9	observed for SiW12/Al catalyst that could be attributed to a strong W-
	catalysts. This decomposition allows to obtain the relative amounts	O bond compared to the Mo-O one that could delay the formation of
	of the different Mo and W species that are gathered in Table 7. The	WS2 phase compared to that of MoS2 phase at a given sulfidation
	sulfidation rate corresponding to the MoS2 atomic % is very high	temperature 50,51 . The presence of Mo in catalyst formulation clearly
	(above 80 %) in the three Mo containing catalysts, compared with	leads to increase W sulfidation that reaches 61 and 67 % for
	sulfidation rate in the literature for conventional HDS	SiMo3W9/Al and Ref Mo3W9 respectively.
	catalysts (around 60 %) 49 . It shows that the molybdenum content

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Acknowledgements

Chevreul Institute (FR 2638), Ministère de l'Enseignement Supérieur et de la Recherche, Région Nord -Pas de Calais and FEDER are acknowledged for supporting and funding partially this work. This work was supported by a public grant overseen by the French National Research Agency(ANR) as part of the "Investissements d'Avenir" program (reference: ANR-10-EQPX-45) as well as the Kolmogorov PHC (project number 38175WC). The work was also supported the Ministry of Education and Science of the RF under project No. 14.586.21.0054 (project unique identifier RFMEFI58617X0054). M. Nikulshina thanks French Embassy in Russia for the Vernadsky fellowship and Haldor Topsøe Company for the grant to perform PhD thesis.

ARTICLE

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Please do not adjust margins Please do not adjust margins (mol h -1 g -1 )

Naphthalene Hyd (%)

Rate constant kHyd ×10 -5 (mol h -1 g -1 )

Conflicts of interest

There are no conflicts to declare. Supporting Information Available: Intra HPA cation-anion bond distances. Bulk H4[SiMo1W11O40] and H4[SiMo3W9O40] EXAFS oscillations at the Mo K and at the W LIII edges: FIT and experimental data. X-Ray crystallographic files (CIF). Bulk H4[SiMo1W11O40], H4[SiMo3W9O40], H4[SiMo12O40] and H4[SiW12O40] IR and Raman spectra and TGA-DSC analysis.

Notes and references