Vincent Escande

Eddy Petit

Laetitia Garoux

Clotilde Boulanger

Claude Grison

Switchable Alkene Epoxidation/Oxidative Cleavage with H 2 O 2 /NaHCO 3 : Efficient Heterogeneous Catalysis Derived from Biosourced Eco-Mn

Keywords: Alkene epoxidation, Alkene oxidative cleavage, Heterogeneous catalyst, Polymetallic catalysis, Ecocatalysis, Hyperaccumulating plants, Phytoextraction

A novel Mn-derived catalyst was prepared starting from the biomass of Mn-hyperaccumulating plants growing on metal-rich soils. Recovery of this biomass as valueadded "ecocatalyst" provides incentives for the development of phytoextraction programs on soils degraded by mining activities. Characterization of the resulting plant-based catalyst Eco-Mn by inductively coupled plasma mass spectrometry (ICP-MS), X-ray diffraction (XRD), X-ray fluorescence spectrometry (XRF), and X-ray photoelectron spectroscopy (XPS) demonstrated the presence of unusual polymetallic complexes of Mn(II) in the catalyst, along with Fe(III). Incorporation of these species into montmorillonite K10 as solid support provided a supported Eco-Mn catalyst, whose properties were investigated for alkene epoxidation with H 2 O 2 (30 wt %)/NaHCO 3 (0.2 M) as a green terminal-oxidizing reagent. The supported Eco-Mn catalyst demonstrated a high efficiency for styrene epoxidation, with only 0.31 mol % Mn, a much lower content of Mn than in previously described Mn-derived heterogeneous catalysts. Although Fe was also present in the supported Eco-Mn catalyst, comparison experiments showed that Fe had only a limited role in the catalysis. The water content in the reaction medium had a beneficial effect, increasing the reaction efficiency. The supported Eco-Mn catalyst was recycled four times without any loss of activity. Comparison of its properties to those of heterogeneous catalysts made by incorporation of commercial MnCl 2 •4H 2 O and FeCl 3 •6H 2 O highlighted the superior catalytic activity of polymetallic species present in the biosourced catalyst. The substrate scope of the method was extended to various alkenes, including bulky natural products which were epoxidized with high yields (up to 99%), sometimes much higher than those obtained with already described Mn-derived heterogeneous catalysts. Finally, by simple adjustments of reaction conditions, the method allowed controlled access to aldehydes by oxidative cleavage of various styrene-derived substrates (up to 93% yield). The method thus constitutes a valuable alternative not only to classical epoxidation reagents but also to oxidative cleavage of styrene-derived molecules, which usually involves toxic and hazardous reagents.

INTRODUCTION

Epoxides are key building blocks in organic chemistry, notably used for the synthesis of complex natural molecules or as intermediates in the preparation of important commercial products. [START_REF] Yudin | Aziridines and Epoxides in Organic Synthesis[END_REF][START_REF] Das | Heterocycles in Natural Product Synthesis[END_REF][START_REF] Xia | Advances in homogeneous and heterogeneous catalytic asymmetric epoxidation[END_REF] Olefin epoxidation is one of the main processes by which these compounds are prepared. Classical methods rely for the most part on the use of organic peroxides and peracids, such as m-chloroperoxybenzoic acid (m-CPBA). [START_REF] Sienel | Ullmann's Encyclopedia of Industrial Chemistry[END_REF] However, the use of these epoxidation reagents in industrial processes is called into question by recent legislation [START_REF] Wilson | Toward a New U.S. Chemicals Policy: Rebuilding the Foundation to Advance New Science, Green Chemistry, and Environmental Health[END_REF][START_REF] Lahl | REACHThe New European Chemicals Law[END_REF] because their handling is associated with safety issues and produces large amounts of wastes. [START_REF] Adams | Development of GSK's reagent guides -embedding sustainability into reagent selection[END_REF] The employment of hydrogen peroxide as terminal oxidant for olefin epoxidation is a promising ecofriendly alternative because this reagent is cheap, readily available, safe at usual concentrations (typically ≤30 wt %), and produces only water as byproduct. [START_REF] Grigoropoulou | Recent developments on the epoxidation of alkenes using hydrogen peroxide as an oxidant[END_REF][START_REF] Rao | Hydrogen Peroxide. In Encyclopedia of Reagents for Organic Synthesis[END_REF] However, the reactivity and selectivity of hydrogen peroxide used alone are limited; therefore, additives and catalysts are needed in order to form more reactive intermediate species. Several catalytic systems have been developed, many of which involve transition metals. [START_REF] Joergensen | Transition-Metal-Catalyzed Epoxidations[END_REF][START_REF] Lane | Metal-catalyzed epoxidations of alkenes with hydrogen peroxide[END_REF][START_REF] Arends | Metal-Catalyzed Asymmetric Epoxidations of Terminal Olefins Using Hydrogen Peroxide as the Oxidant[END_REF] Owing to the depletion of metal resources, new catalysts are required that are inexpensive and that are not based on increasingly scarce metals. [START_REF] Izatt | Challenges to achievement of metal sustainability in our high-tech society[END_REF][START_REF] Hunt | Element Recovery and Sustainability[END_REF] Efforts have been made to develop efficient epoxidation catalysts based on Fe and Ti. [START_REF] Schroder | Iron-Catalyzed Epoxidation of Aromatic Olefins and 1,3-Dienes[END_REF][START_REF] Hasan | Iron-catalyzed epoxidation of olefins using hydrogen peroxide[END_REF][START_REF] Sawada | Titanium-Catalyzed Asymmetric Epoxidation of Non-Activated Olefins with Hydrogen Peroxide[END_REF][START_REF] Nur | Phase-Boundary Catalysis of Alkene Epoxidation with Aqueous Hydrogen Peroxide Using Amphiphilic Zeolite Particles Loaded with Titanium Oxide[END_REF][START_REF] Fraile | Optimization of cyclohexene epoxidation with dilute hydrogen peroxide and silicasupported titanium catalysts[END_REF][START_REF] Timofeeva | Titanium and cerium-containing mesoporous silicate materials as catalysts for oxidative cleavage of cyclohexene with H2O2: A comparative study of catalytic activity and stability[END_REF][START_REF] Guidotti | The use of H2O2 over titaniumgrafted mesoporous silica catalysts: a step further towards sustainable epoxidation[END_REF] Mn is also a good candidate because it is the third most abundant transition metal in the Earth's crust and is considered to be nontoxic. [START_REF] Greenwood | Chemistry of the Elements[END_REF][START_REF] Gerber | Carcinogenicity, mutagenicity and teratogenicity of manganese compounds[END_REF] Mn is involved in several bio logical oxidations, which have inspired the conception of biomimetic oxidation catalysts such as porphyrin-Mn, salen-Mn, phthalocyanin-Mn, and triazamacrocycle-Mn. [START_REF] Que | Biologically inspired oxidation catalysis[END_REF][START_REF] Browne | Modern Oxidation Methods[END_REF] In spite of the efficiency of these catalysts, their preparation is often difficult and low-yielding. Moreover, because of the homogeneous nature of such catalysts, their loss at the end of the reaction increases the cost and the environmental impact of the process. Heterogeneous epoxidation catalysts based on Mn have thus been designed by either grafting of the previous homogeneous catalysts on a solid support or immobilization of Mn derivatives into porous solids. [START_REF] De Vos | Practical heterogeneous catalysts for epoxide production[END_REF] Incorporation of simple Mn salts, readily available and cheap, through solid supports such as zeolites, MCM-41, and graphene oxide has been the object of recent interesting work, although the activity of these catalysts proved to be limited to simple and reactive substrates. [START_REF] Qi | Catalytic epoxidation of alkenes with 30% H2O2 over Mn2+-exchanged zeolites[END_REF][START_REF] Qi | Comparison of different prepared Mn-MCM-41 catalysts in the catalytic epoxidation of alkenes with 30% H2O2[END_REF][START_REF] Zheng | 2+/graphene oxide nanocomposite efficiently catalyzes the epoxidation of alkenes with H2O2[END_REF] Ecocatalysis is a recently established area of sustainable catalysis initiated by our group. [START_REF] Escande | Ecological catalysis and phytoextraction: Symbiosis for future[END_REF][START_REF] Losfeld | Zinc hyperaccumulating plants as renewable resources for the chlorination process of alcohols[END_REF] Taking advantage of the capacity of certain plants to accumulate metallic elements in their cells, [START_REF] Van Der Ent | Hyperaccumulators of metal and metalloid trace elements: Facts and fiction[END_REF][START_REF] Reeves | Phytoremediation of Metal-Contaminated Soils[END_REF][START_REF] Bhargava | Approaches for enhanced phytoextraction of heavy metals[END_REF] the concept consists of the production of biosourced polymetallic catalysts, starting from biomass naturally enriched in trace metals. [START_REF] Grison | Use of metal-accumulating plants for the preparation of catalysts that can be used in chemical reactions[END_REF][START_REF] Grison | Use of certain metal-accumulating plants for implementing organic chemistry reactions[END_REF][START_REF] Grison | Use of certain manganese accumulating plants for the implementation of organic chemistry reactions[END_REF] Crops of metal-hyperaccumulating plants are developed on sites polluted by metallic elements generated by mining activities. Recovery of the resulting biomass, naturally enriched in trace metals, allows a progressive depollution of the contaminated soils. This process, named phytoextraction, thus allows the recycling of metallic elements that are present in soils in concentrations too low to permit recovery by classical mining processes but high enough to be a source of pollution. Thanks to physiological mechanisms of metal-hyperaccumulating plants, the metallic elements are concentrated in their cells with a particular selectivity, specific to each species of plant used. [START_REF] Memon | Implications of metal accumulation mechanisms to phytoremediation[END_REF]39 The resulting biomass thus displays an unusual polymetallic composition. Ecocatalysis processes developed by our group consist of producing polymetallic catalysts (named ecocatalysts, "Eco-M", where M is the metal predominantly accumulated by the plant) starting from this biomass. Thanks to their original structure, these ecocatalysts prove to be highly efficient in various chemical reactions, often leading to selectivity and yield higher than classical catalysts. [40][START_REF] Escande | Preparation of ecological catalysts derived from Zn hyperaccumulating plants and their catalytic activity in Diels-Alder reaction[END_REF][START_REF] Escande | Phytoextracted mining wastes for ecocatalysis: Eco-Mn®, an efficient and eco-friendly plant-based catalyst for reductive amination of ketones[END_REF] In this article, we describe the design and the performances of a heterogeneous catalyst produced from plants from New Caledonia that predominantly accumulate Mn in their leaves. This supported Eco-Mn catalyst demonstrates a high efficiency in the epoxidation of various olefins, including bulky natural molecules, giving yields higher than those obtained with previously described Mn-based heterogeneous catalysts. Moreover, the supported Eco-Mn catalyst also allows a selective access to aldehydes from the starting olefins by simple modification of temperature and reaction time. The methodology thus constitutes a valuable alternative to not only classical methods of epoxidation but also to oxidative cleavage of olefins, which usually involves toxic and polluting reagents. [START_REF] Lee | In Comprehensive Organic Synthesis: Selectivity, Strategy, and Efficiency in Modern Organic Chemistry[END_REF]

EXPERIMENTAL SECTION

Chemicals. Reagents and solvents were purchased from Sigma-Aldrich and were used without further purification.

Preparation of Eco-Mn Catalyst. Leaves of the Mn-hyperaccumulating plant Grevillea exul subsp. rubiginosa (Proteaceae) were harvested before flowering in the Southern Province of the subtropical Pacific island of New Caledonia from homogeneous plant populations. G. exul subsp. rubiginosa was collected at Col de Mouriange, near Le Mont-Dore. The leaves were air-dried and ground. The obtained solid (50 g) was calcined at 400 °C for 5 h, and the resulting powder (1.5 g) was added to 20 mL of a dilute solution of HCl (1 M). The solution was stirred for 2 h at 60 °C. During the heating, the color of the mixture changed from black to dark green. The reaction mixture was then filtered on a pad of Celite. The resulting dark-green solution, composed of different metal chlorides, was concentrated under vacuum, yielding the Eco-Mn catalyst (2.25 g). The resulting Eco-Mn catalyst was stored in a desiccator under vacuum. Purification steps are not mandatory in our process.

Incorporation of Eco-Mn Catalyst into Montmorillonite K10. A 500 mg amount of montmorillonite K10 was added to a solution of Eco-Mn catalyst (250 mg in 10 mL of water) and stirred at 90 °C for 8 h. The solid was then recovered by filtration and washed with deionized water (5 × 10 mL) until the disappearance of free Cl -ions in the solution was achieved (negative test with AgNO 3 ). The resulting supported Eco-Mn catalyst was dried in an oven at 120 °C for 3 h, then stored in a desiccator under vacuum.

Characterization of the Catalysts. Inductively coupled plasma mass spectrometry (ICP-MS) analyses were carried out using the metal analysis of total dissolved solutes in water. The samples were diluted to 0.005 g•L -1 with nitric acid (2.5%) and stirred for 30 min until complete dissolution. Analyses of insoluble supported catalysts were carried out after extraction of their metal content by stirring 50 mg of supported catalyst in 5 mL of a 0.1 M solution of ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA disodium salt) for 24 h at 25 °C followed by filtration, according to a procedure adapted from that of Farrah and Pickering. [START_REF] Farrah | Extraction of heavy metal ions sorbed on clays[END_REF] Analyses were carried out on a HR-ICP-MS Thermo Scientific Element XR. Three blanks were recorded for each step of the dilution procedure. Three analyses were carried out for each sample in order to determine the standard deviation of the measurement.

X-ray diffraction (XRD) analyses were carried out on samples dried at 110 °C for 2 h by using a Bruker diffractometer (D8 advance, with a Cu Kα radiation λ = 1.54086 Å) equipped with a Lynxeyes detector.

X-ray fluorescence spectrometry (XRF) analyses were carried out using a Bruker AXS S4 Explorer wavelength-dispersive spectrometer. The quantitative analysis of major and expected elements was carried out on beaded samples to overcome problems of particle size variation as well as mineralogy effects. The powdered samples were mixed with a Li 2 B 4 O 7 flux with a flux/sample ratio equal to 8, heated in a crucible between 400 and 600 °C,and then cast in a platinum dish to produce a homogeneous, glasslike bead.

X-ray photoelectron spectroscopy (XPS) analyses were carried out with an ESCALAB 250 X-ray photoelectron spectrometer from Thermo Electron. The spectra were generated by Al Kα X-ray (1486.6 eV). Each sample, under the form of powder, was mounted on a support holder using double-sided conducting adhesive tape. The surface of the sample that is analyzed has a diameter of 400 μm. All the binding energies were referenced to the C 1s core-level energy at 284.8 eV.

Analysis of Reaction Products. NMR spectra were recorded on a Bruker Avance 300 spectrometer at room temperature. [START_REF] Yudin | Aziridines and Epoxides in Organic Synthesis[END_REF] H frequency is 300 MHz; [START_REF] Izatt | Challenges to achievement of metal sustainability in our high-tech society[END_REF] C frequency is 75 MHz. IR spectra were recorded on a PerkinElmer Spectrum 100 FT-IR spectrometer in ATR mode. GC-MS analyses were carried out on a Shimadzu QP2010SE apparatus equipped with a 30 m × 0.25 mm × 0.25 μm ZB-5MSi Guardian column (Phenomenex) with hydrogen as carrier gas.

Representative Procedure for Epoxidation of Alkenes. In a typical procedure, styrene (28.7 μL, 0.25 mmol) was added to a mixture of DMF (1 mL) and water (0.6 mL) in a 5 mL reaction vial equipped with a magnetic stirring bar. The mixture was stirred and cooled to 0 °C in an ice bath; then, supported Eco-Mn catalyst was added (39 mg, Mn 0.31 mol %, Fe 1.82 mol %). A freshly prepared mixture of H 2 O 2 (commercial 30 wt % solution, 85 μL, 0.8 mmol) and aqueous NaHCO 3 (0.2 M solution, 250 μL, 0.05 mmol) was then added under stirring to the cooled styrene solution dropwise over 30 min. After 4 h of reaction at 0 °C, the catalyst was filtered off; the reaction mixture was extracted with diethyl ether and then quantitatively analyzed by GC-MS using biphenyl as internal standard. The recovered catalyst was washed with water (2 × 5 mL) then acetone (2 × 5 mL) and dried in an oven at 110 °C for 4 h before the next run.

Representative Procedure for Oxidative Cleavage of Alkenes. In a typical procedure, styrene (28.7 μL, 0.25 mmol) was added to a mixture of DMF (1 mL) and water (0.6 mL) in a 5 mL reaction vial equipped with a magnetic stirring bar. The mixture was stirred and cooled to 0 °C in an ice bath; then, supported Eco-Mn catalyst was added (39 mg, Mn 0.31 mol %, Fe 1.82 mol %). A freshly prepared mixture of H 2 O 2 (commercial 30 wt % solution, 318.8 μL, 3.0 mmol) and aqueous NaHCO 3 (0.2 M solution, 250 μL, 0.05 mmol) was then added under stirring to the cooled styrene solution dropwise over 30 min. After 4 h of reaction at 0 °C, the mixture was heated to 80 °C. After 24 h of heating, the catalyst was filtered off; the reaction mixture was extracted with diethyl ether and then quantitatively analyzed by GC-MS using biphenyl as internal standard.

RESULTS AND DISCUSSION

Preparation and Characterization of the Catalysts. Eco-Mn catalyst was prepared by extraction of the metal content of leaves of the Mn-hyperaccumulating plant G. exul subsp. rubiginosa. The dried leaves were calcined in air in order to eliminate organic matter. Extraction of the resulting powder was then carried out by treatment with a dilute solution (1 M) of hydrochloric acid then filtration, yielding the metallic elements under the form of soluble chlorides. Polymetallic complexes were then recovered in solid form after concentration of the solution, giving the Eco-Mn catalyst without supplementary purification.

The metal composition of the Eco-Mn catalyst was investigated by ICP-MS after dilution of the solid in an aqueous solution of 2.5% nitric acid (Table 1).

The analysis showed that Mn was the major transition metal in the ecocatalyst (6.72 wt %). Small amounts of Fe and Ni were also observed. Mg, Ca, and Al were also present because they are essential for plant growth. Because of the natural origin of the Eco-Mn catalyst, some questions may arise concerning the reproducibility of the metal content of the ecocatalyst. The reproducibility of similar ecocatalysts was studied in a previous article, which showed that the levels of transition elements are only slightly variable between samples derived from different plants of the same species. 40 The Eco-Mn catalyst was prepared from several batches of G. exul subsp. rubiginosa in order to homogenize the content and minimize possible variations in the composition.

XRD analyses were carried out in order to determine the structure of the polymetallic complexes in the Eco-Mn catalyst (Figure 1 and Table 2). Two polymetallic chlorides were detected: CaMg 2 Cl 6 (H 2 O) 12 and KMnCl 3 , which was a masked form of MnCl 2 . Manganese was thus present in Eco-Mn under the oxidation state Mn(II). XRD did not reveal the presence of molecules containing Fe or Al, which were detected by elemental analysis. This was probably due to the noncrystalline nature of these species within the Eco-Mn catalyst.

XPS analyses confirmed that Mn was present under the oxidation state Mn(II) in the Eco-Mn catalyst (Figure 2i) because the spectrum displayed a peak for Mn 2p 3/2 at 641.8 eV, with a satellite peak at 647.4 eV. This configuration has been described as characteristic of the oxidation state (+II) for Mn. [START_REF] Nesbitt | Interpretation of XPS Mn(2p) spectra of Mn oxyhydroxides and constraints on the mechanism of MnO2 precipitation[END_REF] A similar pattern was observed for Fe (Figure 2ii), with the presence of a peak for Fe 2p 3/2 at 711.7 eV, accompanied by a satellite peak at 725.3 eV, which is characteristic of Fe(III). [START_REF] Grosvenor | Investigation of multiplet splitting of Fe 2p XPS spectra and bonding in iron compounds[END_REF] This oxidation state for Fe was confirmed by a simple colorimetric test with ammonium thiocyanate, showing an intense red coloration upon addition of Eco-Mn, characteristic of Fe(III) thiocyanate formation. [START_REF] Feigl | Spot Tests in Inorganic Analysis[END_REF] To ensure the recovery of the Eco-Mn catalyst at the end of the epoxidation reaction, we used montmorillonite K10 as solid support. Previous works have described the preparation and activity of heterogeneous catalysts for epoxidation made by incorporation of Mn(II) simple salts into other solid supports, such as silica, [START_REF] Qi | Catalytic epoxidation of alkenes with 30% H2O2 over Mn2+-exchanged zeolites[END_REF] siliceous mesoporous material TUD-1, [START_REF] Ramanathan | Characterization and Catalytic Properties of the Novel Manganese-Containing Amorphous Mesoporous Material MnTUD-1[END_REF] zeolites Na-Y, Na-beta, Na-ZSM-5, Na-4 Å, [START_REF] Qi | Catalytic epoxidation of alkenes with 30% H2O2 over Mn2+-exchanged zeolites[END_REF] MCM-41, [START_REF] Qi | Comparison of different prepared Mn-MCM-41 catalysts in the catalytic epoxidation of alkenes with 30% H2O2[END_REF] or graphene oxide. [START_REF] Zheng | 2+/graphene oxide nanocomposite efficiently catalyzes the epoxidation of alkenes with H2O2[END_REF] Insoluble manganese oxides have also been used as heterogeneous catalysts. [START_REF] Qi | Selective epoxidation of alkenes with hydrogen peroxide over efficient and recyclable manganese oxides[END_REF] Because montmorillonite K10 is often used in the preparation of cation-exchanged heterogeneous catalysts, [START_REF] Corneĺis | In Encyclopedia of Reagents for Organic Synthesis[END_REF] we decided to investigate the properties of a montmorillonite-K10-supported Eco-Mn catalyst, prepared following an already-described impregnation procedure. [START_REF] Qi | Catalytic epoxidation of alkenes with 30% H2O2 over Mn2+-exchanged zeolites[END_REF] The metal content of the supported Eco-Mn catalyst was determined by XRF (Table 3).

The Mn content in the supported Eco-Mn catalyst was only 0.11 wt %. In the same manner, all the other metals were present in less than 1.00 wt %, except for Al (6.00 wt %), which is a natural constituent of montmorillonite K10 and whose presence is not the result of the incorporation of elements from Eco-Mn. Mn and Fe contents were confirmed by ICP-MS analysis after EDTA extraction of the supported Eco-Mn catalyst. It should be noted that except for Mn (and Ni in a lesser extent), all the metal contents are lower in supported Eco-Mn than in pristine K10, a difference which is particularly marked for Fe. Such leaching of metals originally present in montmorillonite K10 during incorporation of Eco-Mn could be due to the slightly acidic dissolution of Eco-Mn in water (observed pH was 4), which is a result of the hydrolysis of metal chlorides present in Eco-Mn.

Owing to the very low content of Mn in the supported Eco-Mn catalyst, it was not possible to observe clearly the corresponding signals in XRD and XPS analyses. Acquired spectra were not significantly different from those of the solid support alone. Only a weak signal related to a Mn species was detected by XRD (Figure S2). This was attributed to a potassium manganese oxide K 3 (MnO 4 ) 2 previously described particularly as a product of thermal decomposition of KMnO 4 . [START_REF] Boldyrev | On the question of the existence of K3(MnO4)2 as an intermediate phase in the thermal decomposition of potassium permanganate[END_REF][START_REF] Herbstein | Old and new studies of the thermal decomposition of potassium permanganate[END_REF][START_REF] Cannon | Electronic Interaction in an Outer-Sphere Mixed-Valence Double Salt: A Polarized Neutron Diffraction Study of K3(MnO4)2[END_REF] However, because of the weakness of the signal, this presence remains a hypothesis to be confirmed. We decided to investigate the catalytic performances of the supported Eco-Mn catalyst in order to determine if its low Mn content was sufficient to promote the epoxidation of alkenes.

Optimization of Reaction Conditions. We selected reaction conditions of epoxidation that entail the formation, as epoxidizing species, of peroxymonocarbonate (HCO 4 -) produced in situ by mixing hydrogen peroxide and aqueous sodium hydrogen carbonate. [START_REF] Bakhmutova-Albert | Kinetics and Mechanism of Peroxymonocarbonate Formation[END_REF] Because these reagents are readily available, cheap, and do not lead to formation of toxic wastes, their use in alkene epoxidation has been studied by several groups. [START_REF] Yao | Epoxidation of alkenes with bicarbonate-activated hydrogen peroxide[END_REF][START_REF] Frank | Surprising stereoselectivity in the Payne epoxidation of terpinen-4-ol with acetonitrile/hydrogen peroxide[END_REF] Although these reaction conditions lead to high yields of epoxides, long reaction times (up to 48 h) were required under the original conditions. Moreover, these experimental procedures often make use of environmentally unfriendly solvents like DMF. Lane and Burgess discovered that the reaction could be accelerated by catalysis with Mn(II) simple salts and that DMF could be replaced by t-BuOH. [START_REF] Lane | A cheap, catalytic, scalable, and environmentally benign method for alkene epoxidations[END_REF][START_REF] Lane | Manganesecatalyzed epoxidations of alkenes in bicarbonate solutions[END_REF] The authors formulated several hypotheses about the role of Mn(II) ions in the catalysis of the reaction, which are still the subject of mechanistic studies. [START_REF] Quinonero | Computational insights to the mechanism of alkene epoxidation by manganese-based catalysts in the presence of bicarbonate[END_REF][START_REF] Saisaha | Mechanisms in manganese catalysed oxidation of alkenes with H2O2[END_REF] These conditions have been adapted by Qi et al. to the reaction with Mn(II)-exchanged supports. [START_REF] Qi | Catalytic epoxidation of alkenes with 30% H2O2 over Mn2+-exchanged zeolites[END_REF] We started our investigation with a test reaction on styrene 1a under these reaction conditions in order to determine the activity of the supported Eco-Mn catalyst. This catalytic activity was compared to those of the sole montmorillonite K10 and of the nonsupported Eco-Mn (Table 4).

We studied the reaction with an equal mass of the three catalysts: The supported Eco-Mn catalyst shows some efficiency with only 0.31 mol % Mn and 1.82 mol % Fe because styrene epoxide 2a was obtained with 41% yield and 80% selectivity (Table 4, entry 1). Benzaldehyde was the only other product formed. The comparison with montmorillonite K10 proved that the support alone could not catalyze the reaction (entry 2). In the same manner, the nonsupported Eco-Mn was inefficient, even with a high loading of Mn (19.57 mol %) and Fe (2.58 mol %) because only 1% conversion was observed (entry 3); the Mn and Fe proportions were imposed by the composition of the Eco-Mn catalyst. The absence of reaction with nonsupported Eco-Mn The influence of the amount of supported Eco-Mn catalyst was then investigated (Figure 3). Conversion of styrene as well as yield and selectivity toward styrene epoxide followed the same trend, increasing progressively until an optimum of 72 mg was reached for supported Eco-Mn catalyst, corresponding to 0.58 mol % Mn and 3.35 mol % Fe. Beyond this limit, although selectivity remained quite stable, conversion and yield dropped dramatically, owing perhaps to an accelerated decomposition of the oxidizing mixture of H 2 O 2 and NaHCO 3 , as observed with high loading of nonsupported Eco-Mn (Table 4, entry 3). A good yield of styrene epoxide (85%) was thus obtained with low loadings of Mn (0.58 mol %) and Fe (3.35 mol %), confirming the efficiency of the supported Eco-Mn catalyst in the reaction.

Solvents other than DMF were tested in order to determine if a greener alternative could be used (Table 5). Previous studies of epoxidation with Mn(II)-supported catalysts found that DMF was the best solvent for the reaction, although t-BuOH was also a possible choice for reactions carried out at room temperature. [START_REF] Lane | A cheap, catalytic, scalable, and environmentally benign method for alkene epoxidations[END_REF][START_REF] Lane | Manganesecatalyzed epoxidations of alkenes in bicarbonate solutions[END_REF] Because under our conditions the reaction was slightly exothermic, we decided to conduct it at 0 °C, excluding the possibility of using t-BuOH, which is solid at this temperature. 4-Methyl-2-pentanol, still liquid at 0 °C, was tested as an alternative to t-BuOH.

DMF remained effectively the best solvent for the reaction because all the other solvents tested led to a conversion less than 20%. Even with 1,2-propylene carbonate, which has been described as a green alternative to DMF because of its similar solvation properties, [START_REF] Schaffner | Organic Carbonates as Solvents in Synthesis and Catalysis[END_REF] only 4% conversion was obtained (Table 5, entry 3). We thus decided to use DMF, but an incidental observation led us to diminish its content in the reaction medium. Indeed, we observed that when we used supported Eco-Mn catalyst that was not perfectly dried the yield of styrene epoxide was slightly better than with anhydrous catalyst. Suspecting an effect due to traces of water, we thus decided to investigate the influence of a controlled addition of water in the reaction medium (Figure 4). To see the effect of this addition, we started with a lower quantity of supported Eco-Mn catalyst than the previously optimized amount. Although without initial water 39 mg of supported Eco-Mn catalyst for 0.25 mmol of styrene (Mn 0.31 mol %, Fe 1.82 mol %) gave 59% conversion, addition of 10 vol % water caused an increase to 91% conversion. An optimum was reached at 30 vol % water, with quantitative conversion of styrene and 91% yield of styrene epoxide. Selectivity increased equally with the addition of water. Beyond 30 vol % water, conversion dropped because of the insolubility of styrene in the solvent mixture. The positive effect of water on reaction efficiency was quite surprising, but Richardson et al. [START_REF] Richardson | Equilibria, kinetics, and mechanism in the bicarbonate activation of hydrogen peroxide: Oxidation of sulfides by peroxymonocarbonate[END_REF] mentioned a similar observation when a H 2 O 2 /NaHCO 3 mixture was used for the oxidation of sulfides. According to those authors, addition of water led to an increase in conversion because of the better solubility of the oxidizing peroxymonocarbonate HCO 4

-in moist solvents. A similar effect allowed us to obtain a quantitative conversion of styrene with a reduced amount of supported Eco-Mn catalyst (Mn 0.31 mol %; Fe 1.82 mol %), while lowering the content of environmentally unfriendly DMF in the total volume of solvent. Addition of water in the other tested solvents (Table 5) did not give significant improvements mainly because of the insolubility of styrene in these solvent mixtures.

Even if water addition allowed a reduction in catalyst amount from 72 to 39 mg for 0.25 mmol of styrene, the mass ratio remains quite high. This is due to the highly diluted character of the active species in the supported Eco-Mn catalyst, which contains only 0.11 wt % Mn. The mineral matrix, composed of the solid support and other elements from Eco-Mn catalyst, represents an important volume but allows an efficient recycling of the catalyst (see below). From the perspective of an application of the procedure on a large scale, the reaction medium remains fluid even with this mass ratio of catalyst versus substrate. Agitation is not a problem, and filtration is easy.

The importance of slow dropping of H 2 O 2 in epoxidation reactions has been underlined in previous papers, demonstrating its impact on the selectivity of the reaction and on H 2 O 2 decomposition. [START_REF] Fraile | Optimization of cyclohexene epoxidation with dilute hydrogen peroxide and silicasupported titanium catalysts[END_REF][START_REF] Timofeeva | Titanium and cerium-containing mesoporous silicate materials as catalysts for oxidative cleavage of cyclohexene with H2O2: A comparative study of catalytic activity and stability[END_REF][START_REF] Guidotti | The use of H2O2 over titaniumgrafted mesoporous silica catalysts: a step further towards sustainable epoxidation[END_REF] The amount of residual H 2 O 2 was thus measured (titration by manganimetry, [START_REF] Kolthoff | In Volumetric Analysis[END_REF] Recycling and Leaching Studies. Using these new reaction conditions, we studied the recycling of the supported Eco-Mn catalyst and its possible leaching. The recycling was carried out four times, after filtration, washing, and then drying of the supported Eco-Mn catalyst (Table 6).

Conversion and yield remained unchanged after the fourth recycling, indicating high stability of the supported Eco-Mn catalyst, even in the oxidizing conditions of the reaction. Starting with a content of 0.11 wt % Mn, a small decrease was first observed; then, the value stayed stable around 0.080 wt % over the following four runs. The Fe content, initially 0.65 wt %, remained virtually unchanged even after the fourth recycling, confirming the quasi-absence of leaching of the catalyst, even in the aqueous medium used. These observations were confirmed by a filtration test, carried out at the temperature of the reaction, as described by Sheldon et al. [START_REF] Sheldon | Heterogeneous Catalysts for Liquid-Phase Oxidations: Philosophers' Stones or Trojan Horses?[END_REF] After 30 min of reaction, the catalyst was filtered, and the conversion determined to be 67% (selectivity 91%). The filtrate was allowed to react further, until 4 h of reaction. The resulting conversion was then 80% (selectivity 88%), whereas in a parallel blank test without filtration, conversion reached 98% (selectivity 90%). The significant difference in conversion observed between filtered and nonfiltered reactions demonstrates that although leaching traces of Mn cannot be totally excluded the active species are of heterogeneous character in the supported Eco-Mn catalyst.

Comparison with Supported Commercial Simple Salts of Mn(II) and Fe(III). To determine the origin of the activity of the supported Eco-Mn catalyst, we prepared similar catalysts with montmorillonite-K10-supported commercial salts of Mn(II) and Fe(III). The procedure used for the preparation of the supported Eco-Mn catalyst was followed with MnCl 2 •4H 2 O, FeCl 3 •6H 2 O, and a mixture of these salts. The proportions of salts used were the same as in the starting nonsupported Eco-Mn. Their catalytic activity was then tested under the same conditions as for the supported Eco-Mn catalyst (Table 7).

Reaction with K10-supported MnCl 2 •4H 2 O gave 97% conversion of styrene and 83% yield of styrene epoxide, values similar to those obtained with the supported Eco-Mn catalyst (Table 7, entries 1 and 2). Supported FeCl 3 •6H 2 O led to an almost absence of reaction, with only 5% conversion of styrene (entry 3). These results proved that the activity of the supported Eco-Mn catalyst was effectively due to Mn(II) species, whereas Fe(III) had only a very limited role in the catalysis. A third catalyst, made with incorporation of a mixture of MnCl 2 •4H 2 O and FeCl 3 •6H 2 O in montmorillonite K10, was tested to investigate a possible synergistic effect of the metallic elements (entry 4). Conversion (98%), yield (86%), and selectivity (88%) were slightly better with this catalyst than with the supported MnCl 2 •4H 2 O, confirming the interest of the polymetallic catalysis. However, these values were lower than those obtained with the supported Eco-Mn catalyst, proving that the efficiency of this biosourced catalyst was due to particular complexes of the metallic elements accumulated in the starting biomass. The use of supported Eco-Mn catalyst is thus beneficial in terms of efficiency in two ways: First, it displays a superior activity in styrene epoxidation than MnCl 2 •4H 2 O. Second, Eco-Mn is a more desirable form of the metal because it is reclaimed from polluted soils. Although the process requires energy and time to extract Eco-Mn from the Mn-hyperaccumulating plant, this approach is one of the very few ways of valorization of biomass derived from phytoextraction. [START_REF] Vigil | Is phytoremediation without biomass valorization sustainable?  Comparative LCA of landfilling vs. anaerobic co-digestion[END_REF][START_REF] Ernst | Phytoextraction of mine wastes -Options and impossibilities[END_REF][START_REF] Van Der Ent | Farming for Metals in the Future?[END_REF] In traditional mining operations, ecological rehabilitation of degraded soils is seen only as a negative obligation and constraint. The use of Eco-Mn derived from Mn-hyperaccumulating plants could change this perspective. Ecocatalysis increases the value of this biomass, thus changing the legal obligation of restoration into new scientific and financial opportunities.

Substrate Scope of the Supported Eco-Mn-Catalyzed Epoxidation of Alkenes. On the basis of the above studies, the epoxidation of further alkenes was carried out to determine the substrate scope of the method (Table 8). In some cases, the alkenes were not soluble in the DMF/water 7:3 mixture, resulting in poor conversion. When necessary, the percentage of water was therefore reduced. For all the tested substrates, the beneficial effect of water was confirmed, providing that the alkene remained soluble in the solvent mixture. For cases where the tested alkene was not soluble, the addition of a surfactant agent (sodium dodecyl sulfate) was examined but did not significantly improve the conversion.

A first test with norbornene 1b confirmed the efficiency of the method, giving 99% yield of norbornene epoxide 2b (Table 8, entry 1). Reactions on (R)-(+)-limonene 1c and citral 1f (entries 2 and 5) indicated that electron-rich double bonds are selectively epoxidized. A moderate activity was noted with (R)-(+)-pulegone 1g, whose C-C double bond is highly deactivated by conjugation with carbonyl (entry 6). The method proved to be efficient even on bulky natural products, such as α-pinene 1d, giving 75% yield of α-pinene epoxide 2d with complete selectivity (entry 3). Previously described Mn(II)zeolite catalysts were much less effective, showing the interest of the supported Eco-Mn catalyst. [START_REF] Qi | Catalytic epoxidation of alkenes with 30% H2O2 over Mn2+-exchanged zeolites[END_REF] According to literature, linalool epoxide rearranges as soon as formed to give a cyclized product (2e) with 63% yield. [START_REF] Klein | Die Chemie der Linalooloxide[END_REF] Fair to good yields of epoxides were obtained on complex and bulky structures, such as nopol 1j (74%, entry 9) and β-caryophyllene 1k (42%, entry 10), with excellent selectivity. However, the method was inefficient on terminal aliphatic alkenes, such as n-octene 1l, as already observed with other Mn(II)-based catalysts (entry 11). [START_REF] Qi | Catalytic epoxidation of alkenes with 30% H2O2 over Mn2+-exchanged zeolites[END_REF][START_REF] Lane | Manganesecatalyzed epoxidations of alkenes in bicarbonate solutions[END_REF] By contrast, indene 1m, cyclooctene 1n, and cyclohexene 1o gave epoxides with much higher yields than previously described with Mn(II)-zeolite catalysts (entries 12-14). Cyclooctene epoxide 2n was thus formed in yield nearly double the values reported with other Mn-derived catalysts. [START_REF] Qi | Catalytic epoxidation of alkenes with 30% H2O2 over Mn2+-exchanged zeolites[END_REF] Extension of the Method to Oxidative Cleavage of Alkenes. During optimization of styrene epoxidation, we always observed the formation of a small amount of benzaldehyde. We tried to determine if it was possible to obtain this aldehyde selectively, starting from the alkene. We started our study with the effect of temperature, by heating the reaction medium to 80 °C instead of cooling to 0 °C, but no reaction was observed because the alkene remained unchanged. In contrast, when we used the previous epoxidation conditions (4 h at 0 °C) and then heated the medium to 80 °C, after formation of the styrene epoxide, a significant increase in benzaldehyde yield was observed. To reach a nearly quantitative yield of benzaldehyde, however, it was necessary to increase reaction time and amount of hydrogen peroxide (Table 9, entries 1-2). Table 8. Substrate Scope of the Supported Eco-Mn-Catalyzed Epoxidation

The presence of 1-phenyl-1,2-ethanediol (28% yield after 10 h, entry 1) and then its disappearance while benzaldehyde was formed in nearly quantitative yield (after 24 h, entry 2) was an indication of the possible mechanism. The epoxide formed initially was probably opened by hydrolysis into a 1,2-diol under catalysis by supported Eco-Mn catalyst. (Without the catalyst, styrene remained unchanged.) The 1,2-diol then underwent an oxidative cleavage into aldehyde. Tests starting directly from styrene epoxide or 1-phenyl-1,2-ethanediol led to nearly quantitative yields of benzaldehyde after 24 h, which confirms the mechanism hypothesis. This oxidative cleavage seemed to be specific to the supported Eco-Mn catalyst because only 24% yield of benzaldehyde was observed with supported commercial MnCl 2 •4H 2 O/FeCl 3 •6H 2 O prepared under the same conditions as the supported Eco-Mn catalyst (entry 3). This reaction with supported commercial MnCl 2 •4H 2 O/FeCl 3 • 6H 2 O produced 71% yield of 1,2-diol, showing that the limiting step was not the formation of 1,2-diol but rather its cleavage. Only the original composition of the supported Eco-Mn catalyst allowed the conversion of styrene into benzaldehyde with 93% yield (entry 2).

Further alkenes were tested to determine the scope and limitations of the reaction. Presence of electron-withdrawing groups on the aromatic ring seemed to be unfavorable because only 11% yield of aldehyde 3c was obtained starting from 3-chlorostyrene 1q (entry 5). Because the corresponding epoxide was however formed in 68% yield, this indicated that the limiting step was the opening of the epoxide. trans-Anethole 1p, bearing both a methyl-enriched C-C double bond and a methoxyenriched aromatic ring, gave a better yield of p-anisaldehyde 3b (51% yield, entry 4).

Reaction with cinnamyl acetate 1r (entry 6) furnished interesting information on the mechanism. After heating, 53% yield of benzaldehyde was obtained, in line with our expectations, but 19% yield of cinnamaldehyde was also observed. Formation of cinnamaldehyde could not be explained by oxidative cleavage of the 1,2-diol after epoxide hydrolysis. Cinnamaldehyde was probably formed by hydrolysis of the acetate into cinnamyl alcohol, which was then oxidized into cinnamaldehyde. Such oxidation of an allylic alcohol is characteristic of the oxidizing activity of Mn(IV), producing aldehydes without further oxidation into carboxylic acid, as is usually observed with most oxidizing reagents. [START_REF] Hirano | Oxidation by chemical manganese dioxide. Part 3. Oxidation of benzylic and allylic alcohols, hydroxyarenes and aminoarenes[END_REF][START_REF] Cahiez | Encyclopedia of Reagents for Organic Synthesis[END_REF] It is thus possible that Mn(II) present in the supported Eco-Mn catalyst was oxidized into Mn(IV) by hydrogen peroxide, the formed Mn(IV) thus being responsible for the cinnamyl alcohol oxidation. Mn(IV) could be the active species responsible for the oxidative cleavage of 1,2-diols into aldehydes under our conditions because this reaction was already reported. [START_REF] Ohloff | Conversion of Vicinal Diols into Dicarbonyl-Compounds by Manganese-Dioxide[END_REF] Oxidation of Mn(II) into Mn(IV) by H 2 O 2 /NaHCO 3 was proposed by Lane et al. [START_REF] Lane | Manganesecatalyzed epoxidations of alkenes in bicarbonate solutions[END_REF] The residual amount of H 2 O 2 at the end of the reaction was 0.25 mmol, which corresponds to 75 mol % decomposition of the initial amount of H 2 O 2 (in the hypothesis of oxidative cleavage by Mn(IV), consuming one molar equivalent of H 2 O 2 for epoxidation and one more for the cleavage). This high value is in accordance with previous observations of the accelerated decomposition of H 2 O 2 during heating in the presence of catalysts. [START_REF] Fraile | Optimization of cyclohexene epoxidation with dilute hydrogen peroxide and silicasupported titanium catalysts[END_REF][START_REF] Timofeeva | Titanium and cerium-containing mesoporous silicate materials as catalysts for oxidative cleavage of cyclohexene with H2O2: A comparative study of catalytic activity and stability[END_REF][START_REF] Guidotti | The use of H2O2 over titaniumgrafted mesoporous silica catalysts: a step further towards sustainable epoxidation[END_REF] These results thus confirmed the interest of the supported Eco-Mn catalyst, giving selectively epoxides or aldehydes, by simple adjustments of the amount of H 2 O 2 and reaction conditions. Tests on aliphatic alkenes showed that the present reaction conditions are limited to styrene-derived compounds. Further optimization of the oxidative cleavage on a larger set of substrates is in progress.

CONCLUSIONS

Starting from the manganese-hyperaccumulating plant G. exul subsp. rubiginosa, we prepared a heterogeneous Eco-Mn catalyst by incorporation of biosourced polymetallic complexes into montmorillonite K10 as solid support. Characterization by ICP-MS, XRD, XRF, and XPS revealed that Mn(II) and Fe(III) were present in the catalyst. Unusual polymetallic complexes, such as KMnCl 3 , were observed. After reaction conditions were optimized, the supported Eco-Mn catalyst demonstrated a high activity in alkene epoxidation, with only 0.31 mol % Mn, which proved to be the main active species. An interesting effect of the presence of water, which increased the efficiency of the method, was observed. Supported commercial salts of Mn(II) and Fe(III), although demonstrating good activity, were not as active and selective as the supported Eco-Mn catalyst, confirming the interest of polymetallic catalysis by the original species present in the biosourced catalyst. The supported Eco-Mn catalyst was recycled four times with no loss of activity. The scope of the methodology was investigated on a wide range of alkenes, including bulky natural products that were epoxidized in fair to excellent yields (up to 99%). In several cases, the supported Eco-Mn catalyst gave much higher yields of epoxides than previously described Mn-derived heterogeneous catalysts. Finally, by a simple adjustment of reaction conditions, the method provided selective access to aldehydes by oxidative cleavage of the starting alkene. This result proved to be particularly interesting because it may provide an alternative to the use of classical oxidative cleavage reagents, which are often toxic and environmentally hazardous. Further investigation is in progress to identify the active polymetallic species of the Eco-Mn catalyst because there is no doubt that they will continue to display very interesting properties for cutting-edge catalysis.

ASSOCIATED CONTENT

X-ray diffraction spectrum of Eco-Mn, detailed XPS spectra of Eco-Mn, ICP-MS analysis of Eco-Mn, and MS spectra of synthesized compounds. (PDF) Table 9. Oxidative Cleavage of Alkenes with the Modified Procedure a Reaction conditions: alkene (0.25 mmol), H 2 O 2 (30 wt %, 3.0 mmol), NaHCO 3 (0.2 M, 0.05 mmol), catalysts (39 mg, Mn 0.31 mol %, Fe 1.82 mol %), DMF/water 7:3 (2 mL), 0 °C for 4 h, then 80 °C for 10-24 h. b Conversion and yield were determined by GC-MS analysis, with biphenyl as internal standard. Synthesized products were identified by comparison of their spectroscopic data with those previously published. c Ratio of yield to conversion as a percentage. d 1-Phenyl-1,2-ethanediol is obtained as side product with 28% yield. e 1-Phenyl-1,2-ethanediol is obtained as side product with 71% yield. f 3-Chlorostyrene epoxide is formed with 68% yield. g Cinnamaldehyde is obtained as side product with 19% yield.

Figure 1 .

 1 Figure 1. Characterization of Eco-Mn by XRD.

Figure 2 .

 2 Figure 2. XPS analysis of Eco-Mn showing (i) Mn 2p 3/2 peak and (ii) Fe 2p 3/2 peak.

da

  Reaction conditions: styrene (0.25 mmol), H 2 O 2 (30 wt %, 0.8 mmol), NaHCO 3 (0.2 M, 0.05 mmol), catalysts (39 mg), DMF (2 mL), 0 °C, 4 h. b Conversion and yield were determined by GC-MS analysis, with biphenyl as internal standard. c Ratio of yield to conversion as a percentage. d n.d.: not determined.

Figure 3 .

 3 Figure 3. Influence of the amount of supported Eco-Mn catalyst on the conversion, yield of styrene epoxide, and selectivity for this compound. Reaction conditions: styrene (0.25 mmol), H 2 O 2 (30 wt %) (0.8 mmol), NaHCO 3 (0.2 M) (0.05 mmol), DMF (2 mL), 0 °C, 4 h. Selectivity is expressed as the ratio of yield to conversion.

  average of three runs) at the end of the reaction in the proposed conditions. A residual amount of 0.28 mmol of H 2 O 2 was determined, indicating a decomposition of 34 mol % the total load of H 2 O 2 , by disproportionation into O 2 and H 2 O.

Figure 4 .

 4 Figure 4. Effect of the addition of water to the reaction medium. Reaction conditions: styrene (0.25 mmol), H 2 O 2 (30 wt %) (0.8 mmol), NaHCO 3 (0.2 M) (0.05 mmol), supported Eco-Mn catalyst (39 mg, Mn 0.31 mol %, Fe 1.82 mol %), DMF/water (2 mL), 0 °C, 4 h. Selectivity is expressed as the ratio of yield to conversion.

a

  Reaction conditions: styrene (0.25 mmol), H 2 O 2 (30 wt %, 0.8 mmol), NaHCO 3 (0.2 M, 0.05 mmol), recycled supported Eco-Mn catalyst (39 mg, Mn 0.31 mol %, Fe 1.82 mol %), DMF/water 7:3 (2 mL), 0 °C, 4 h. b Conversion and yield were determined by GC-MS analysis, with biphenyl as internal standard. c Ratio of yield to conversion as a percentage. d Determined by ICP-MS after EDTA extraction of a sample of supported Eco-Mn catalyst after reaction.

a

  Reaction conditions: styrene (0.25 mmol), H 2 O 2 (30 wt %, 0.8 mmol), NaHCO 3 (0.2 M, 0.05 mmol), catalysts (39 mg), DMF/water 7:3 (2 mL), 0 °C, 4 h. b Conversion and yield were determined by GC-MS analysis, with biphenyl as internal standard. Average of 4 runs. c Ratio of yield to conversion as a percentage.

a

  Reaction conditions: alkene (0.25 mmol), H 2 O 2 (30 wt %, 0.8 mmol), NaHCO 3 (0.2 M, 0.05 mmol), supported Eco-Mn catalyst (39 mg, Mn 0.31 mol %, Fe 1.82 mol %), DMF/water (2 mL), 0 °C, 4 h. b Conversion and yield were determined by GC-MS analysis, with biphenyl as internal standard. Synthesized products were identified by comparison of their spectroscopic data with those previously published. c Ratio of yield to conversion as a percentage. d n.d.: not determined.

Table 1 .

 1 ICP-MS Analysis of Eco-Mn from Grevillea exul subsp. rubiginosa

metal composition Mg wt % (±sd) Ca wt % (±sd) Al wt % (±sd) Mn wt % (±sd) Fe wt % (±sd) Ni wt % (±sd) G. exul subsp. rubiginosa (dry shoots) 0.24 (±0.0013) 0.24 (±0.0030) 0.017 (±0.00014) 0.40 (±0.0028) 0.049 (±0.00079) 0.011 (±0.000077) Eco-Mn derived from G. exul subsp. rubiginosa 5.26 (±0.086) 10.44 (±0.17) 0.24 (±0.015) 6.72 (±0.037) 0.90 (±0.0075) 0.040 (±0.00039)

Table 2 .

 2 Crystalline Species Present in Eco-Mn and Detected by XRD

	mineral species	formula (simplified)
	calcium magnesium chloride hydrate	CaMg 2 Cl 6 (H 2 O) 12
	calcium sulfate hydrate	CaSO 4 (H 2 O) 0.5
	potassium manganese chloride	KMnCl 3

Table 3 .

 3 Metal Composition of the Supported Eco-Mn Catalyst as Established by XRF be due to the rapid decomposition of the oxidizing mixture of H 2 O 2 and NaHCO 3 because an effervescence was noted upon addition of the catalyst. Chemical analysis of the resulting gas revealed that only O 2 was formed, probably by disproportionation of H 2 O 2 into O 2 and H 2 O. Such effervescence was not observed with the supported Eco-Mn catalyst.

	metal composition	Mg wt % (±sd)	Ca wt % (±sd)	Al wt % (±sd)	Mn wt % (±sd)	Fe wt % (±sd)	Ni wt % (±sd)
	pristine montmorillonite K10	1.04 (±0.071)	0.27 (±0.054)	7.54 (±0.060)	n.d. a	2.17 (±0.032)	n.d. a
	supported Eco-Mn	0.70 (±0.066)	0.20 (±0.0062)	6.00 (±0.045)	0.11 (±0.0029)	0.65 (±0.0045)	0.002 (±0.00034)

a n.d.: not detected. may

Table 4 .

 4 Epoxidation of Styrene with Different Catalysts

	entry	catalyst	amount of Mn (mol %)	amount of Fe (mol %)	conversion (%) a,b	yield (%) b	selectivity (%) c
	1	supported Eco-Mn	0.31	1.82	51	41	80
	2	montmorillonite K10		6.06	0	0	n.d. d
	3	Eco-Mn	19.57	2.58	1	1	n.d.

Table 5 .

 5 Effect of Various Solvents on the Epoxidation of Styrene Catalyzed by Supported Eco-Mn

	entry	solvent	conversion (%) a,b	yield (%) b	selectivity (%) c
	1	DMF	95	85	89
	2	acetonitrile	17	17	100
	3	1,2-propylene carbonate	4	4	100
	4	dichloromethane	0	0	n.d. d
	5	water	4	4	100
	6	isopropanol	6	5	83
	7	4-methyl-2-pentanol	5	4	80
	8	glycerol	6	4	67
	9	ethanol	17	16	94
	10 THF	8	5	63
	11 acetone	18	18	100
	12 2-butanone	5	5	100

a Reaction conditions: styrene (0.25 mmol), H 2 O 2 (30 wt %, 0.8 mmol), NaHCO 3 (0.2 M, 0.05 mmol), supported Eco-Mn catalyst (72 mg, Mn 0.58 mol %, Fe 3.35 mol %), solvent (2 mL), 0 °C, 4 h. b Conversion and yield were determined by GC-MS analysis, with biphenyl as internal standard. c Ratio of yield to conversion as a percentage. d n.d.: not determined.

Table 6 .

 6 Recycling and Leaching Studies of the Supported Eco-Mn Catalyst

	entry	recycling	conversion (%) a,b	yield (%) b	selectivity (%) c	postrun Mn content wt % (±sd) d	postrun Fe content wt % (±sd) d
	1	no. 1	97	89	92	0.087 ± 0.00070	0.64 ± 0.0021
	2	no. 2	97	89	92	0.080 ± 0.0014	0.65 ± 0.0003
	3	no. 3	98	91	93	0.086 ± 0.0021	0.64 ± 0.0015
	4	no. 4	98	90	92	0.079 ± 0.0020	0.66 ± 0.0018

Table 7 .

 7 Comparison of Catalysts Made with Biosourced and Commercial Mn(II) and Fe(III) Salts

ACKNOWLEDGMENTS

We thank the Agence Nationale de la Recherche (ANR, programme 11ECOT 011 01), the Agence de l'Environnement et de la Mai ̂trise de l'Energie (ADEME), the Centre National de la Recherche Scientifique (CNRS), and the Fond Europeén de Developpement Reǵional (FEDER) for financial support. Prof. Doyle McKey is greatly acknowledged for helpful discussions and proofreading the manuscript.

AUTHOR INFORMATION

Corresponding Author *E-mail: claude.grison@cnrs.fr.

Notes

The authors declare no competing financial interest.


Anne-Laure Papa

Lionel Maurizi

David Vandroux

Paul Walker

Nadine Millot
email: nmillot@u-bourgogne.fr

Synthesis of titanate nanotubes directly coated with USPIO in hydrothermal conditions: a new detectable nanocarrier

Keywords: Nanoparticles and nanostructures Titanate nanotubes (TiONts), Ultrasmall SuperParamagnetic Iron Oxide (USPIO), hydrothermal synthesis, MRI detection, nanocarrier

For the first time, titanate nanotubes (TiONts) coated with USPIO (Ultrasmall Superparamagnetic Iron Oxide) without polymer functionalization and directly obtained during TiONts formation is reported. The coating of these tubes was performed directly during the TiONts hydrothermal synthesis. After this treatment, in strongly basic conditions, USPIO particles kept their structure and magnetic properties while their size distribution slightly increased. By coupling zeta potential measurements and TEM observations at different pH, it appeared that interactions between nanotubes and USPIO were not electrostatic. This study presents the new designing of a titanate needle shape nanocomposite capable of being detected by MRI for future biomedical applications.

MANUSCRIPT TEXT

Titanate nanotubes (TiONts) have increasingly gained attention since their discovery [START_REF] Kasuga | Titania nanotubes prepared by chemical processing[END_REF] . They are generally synthesised by a hydrothermal treatment of a TiO2 precursor (150 °C, 5 bar) in strongly basic conditions (NaOH, 10 mol.L -1 ) [START_REF] Papa | Effect of reaction parameters on composition and morphology of titanate nanomaterials[END_REF] . These nanotubes have a tubular morphology being rolled up in a spiral fashion, with an inner cavity: their length can attain several hundred nanometers and their diameter only a few nanometers (3-4 nm < dinner < 10 nm and 10 nm < doutside < 20 nm) [START_REF] Papa | Effect of reaction parameters on composition and morphology of titanate nanomaterials[END_REF] . They also develop large specific surfaces depending on reaction parameters. These particularities induce an increase in the possible interactions with their outside environment.

Nanomedicines have attracted more and more attention over the last decade. Notably, this is reflected in the development of new therapeutics to target, detect and treat pathologies. Titanium oxides have revealed themselves as being promising material for biomedical applications. Indeed, titanium oxide surfaces have already been used as implantable devices [START_REF] Rupp | Enhancing surface free energy and hydrophilicity through chemical modification of microstructured titanium implant surfaces[END_REF] , pointing out the biocompatibility of titanium due to the TiO2 passivation layer spontaneously formed on its surface. Recently, a study evaluated the effective stability of titanium dioxide (TiO2)-blasted dental implants over a 3-year period [START_REF] Veltri | Stability values of titanium dioxide-blasted dental implants in edentulous maxillas: a 3-year pilot study[END_REF] . As for titanate nanotubes (TiONts), they have been used to replace hydroxyapatite in bone regeneration because they promote faster development of osteoblasts [START_REF] Kubota | Titanium oxide nanotubes for bone regeneration[END_REF] . They have also been used as electrochemical biosensors for dopamine detection [START_REF] Niu | Titanate nanotubes: preparation, characterization, and application in the detection of dopamine[END_REF] in the diagnosis of Parkinson's disease.

To our knowledge, titanate nanotubes have not yet been reported in the literature for vectorisation studies in the biomedical field. Nonetheless, reports have already shown that the shape of nanoparticles, used as carriers, affects biodistribution [START_REF] Decuzzi | Size and shape effects in the biodistribution of intravascularly injected particles[END_REF] . It has also been shown previously that elongated shape organic nanoparticles were more easily internalised into cells than their spherical counterparts with similar volumes [START_REF] Gratton | The effect of particle design on cellular internalization pathways[END_REF] . The needle shape of these nanotubes, leading to a high internalisation potential, was the rationale for future studies, with the prospect to evaluate the biodistribution of this new nanocarrier. Several possibilities may allow the detection of titanate nanotubes by imaging modalities: grafting a paramagnetic compound for Magnetic Resonance Imaging (MRI), grafting of radioisotopes for Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT) and grafting of fluorescent dyes. The present paper reports the synthesis, the designing and the characterisation of a new TiONts-USPIO carrier for future studies of drugs vectorisation and MRI detection applications.

Today, nanotubes attract attention in the biomedical field. For example, Calucci et al. published in 2010 a study on boron nitride nanotubes as T2-weighted MRI contrast agents [START_REF] Calucci | Boron Nitride Nanotubes as T2-Weighted MRI Contrast Agents[END_REF] . Additionally, carbon nanotubes (CNTs), have already been tested to deliver therapeutic or detection molecules [START_REF] Mcdevitt | Tumor targeting with antibody-functionalized, radiolabeled carbon nanotubes[END_REF][START_REF] Pantarotto | Translocation of bioactive peptides across cell membranes by carbon nanotubes[END_REF] .

However, they have several major drawbacks: they are insoluble in most solvents [START_REF] Vaisman | The role of surfactants in dispersion of carbon nanotubes[END_REF] and their extremities are closed. In addition, they need to be functionalised to render them more hydrophilic.

Several groups reported CNTs used as MRI contrast agents [START_REF] Al Faraj | In Vivo Imaging of Carbon Nanotube Biodistribution Using Magnetic Resonance Imaging[END_REF] . However, the attribution of susceptibility effects remains controversial. They were due to iron impurities for some [START_REF] Vaisman | The role of surfactants in dispersion of carbon nanotubes[END_REF] , and due to both the iron impurity and the CNTs themselves for others [START_REF] Al Faraj | In Vivo Imaging of Carbon Nanotube Biodistribution Using Magnetic Resonance Imaging[END_REF] . Notice that in most cases, studies published employed SWCNTs functionalized by Gd 3+ chelates [START_REF] Hartman | Gadonanotubes as ultrasensitive pH-smart probes for magnetic resonance imaging[END_REF] to obtain MRI contrast.

Different possibilities were encountered in the literature to obtain coated or doped TiONts. TiONts were typically coated with metal nanoparticles such as Au, Pd, Pt, RuO(OH), Ni and CdS which were used for catalysis [START_REF] Bavykin | Elongated Titanate Nanostructures and Their Applications[END_REF] . Authors assumed that the metal nanoparticles were deposited on both internal and external surfaces of tubes. Previous studies have also described direct hydrothermal synthesis of titanate nanotubes doped with magnetic metal ions (Fe 3+ , Ni 2+ , Mn 2+ , Co 2+ ) [START_REF] Wang | Direct hydrothermal synthesis and magnetic property of titanate nanotubes doped magnetic metal ions[END_REF][START_REF] Bavykin | Elongated Titanate Nanostructures and Their Applications[END_REF] . Some authors have described iron oxide "tube-in-tube" nanostructures [START_REF] Song | A hydrothermal method for preparation of α-Fe2O3 nanotubes and their catalytic performance for thermal decomposition of ammonium perchlorate[END_REF][START_REF] Suber | Synthesis, morphology, and magnetic characterization of iron oxide nanowires and nanotubes[END_REF] . After their hydrothermal synthesis, a thermal treatment allowed to convert hematite nanotubes into maghemite [START_REF] Jia | Single-Crystalline Iron Oxide Nanotubes[END_REF] . Another study described TiO2 nanotubes functionalised by iron oxide nanoparticles [START_REF] Kontos | Self-Organized Anodic TiO2 Nanotube Arrays Functionalized by Iron Oxide Nanoparticles[END_REF] . These tubes were first synthesised by electrochemical anodisation (aligned tubes with much higher dimensions than those obtained by hydrothermal treatment: diameter about 100 nm) and they were then functionalised with iron nanoparticles by dextrin linking. A study deals with iron-coated TiO2 nanotubes and is dedicated to photocatalytic studies [START_REF] An | Iron-coated TiO2 nanotubes and their photocatalytic performance[END_REF] . The coating is obtained in two step: after titanate nanotubes synthesis by treatment with a Fe(OH)3 solution. To the best of our knowledge, there have been no prior studies on TiONts coated with USPIO during the hydrothermal synthesis of nanotubes without polymer linkage between both oxide particles. This is reported for the first time in the present study.

After hydrothermal synthesis, the resulting light brown coloured product, and in particular whitish filaments (Figure 1a), was attracted by magnetization (Figure 1b). This observation leads to two conclusions: i) iron oxide nanoparticles kept their magnetic properties during the 24 hours of hydrothermal treatment, ii) titanates were also attracted and proved a link between nanotubes and magnetic nanoparticles. TEM observations have shown that titanates had tubular morphology (Figure 2) as expected and described for the previously reported synthesis of TiONts 2 . The presence of iron oxide nanoparticles did not disturb a lot the "rolling up" mechanism of nanotubes and no other by-products such as remaining TiO2 particles, titanate nanoribbons or nanosheets were observed. The only difference observed was a diameter distribution of TiONts when USPIO were added during nanotubes synthesis: between 10 nm (Figure 2 a1) and 20 nm (Figure 2 a2) in comparison with mostly 10 nm for the reference synthesis of TiONts (without USPIO) 2 (Figure 2d). A first explanation could be the inclusion c)

of USPIO particles into the TiONts inner cavity during the sheet rolling-up mechanism [START_REF] Zhang | Formation Mechanism of H2Ti3O7 Nanotubes[END_REF] and leading maybe to highest diameters figure 2c and2d. Moreover, heterogeneities of the diameter along a single tube have been observed (Figure 2 a2), such perturbations may be due to USPIO inclusion in the inner cavity of the tube. Nevertheless, it remains difficult to be fully affirmative about this USPIO entrapment only thanks HRTEM investigation. Another explanation could be the influence of USPIO in solution which may affect the surface tension of the titanate sheets and modify nanotube enrollment. Indeed, small changes in synthesis parameters of titanate nanotubes could lead to chemical counterparts having different shapes (as nanowires, nanoribbons) [START_REF] Papa | Effect of reaction parameters on composition and morphology of titanate nanomaterials[END_REF] . So it is not exclude that the introduction of USPIO during tubes synthesis could also disturb the driving forces leading to the own morphology of the tubes (number of layers, diameter, etc…), as here about the diameter variation of the tubes (Figure 2d). Moreover, it is well known that modifications of nanoparticles surface, here nanotubes surface, lead to modifications of surface energy and surface stress, both responsible for structural disturbance. 24 n Concerning USPIO distribution on nanotubes surface, all TiONts carried USPIO, but not in a homogeneous way. Some tubes were abundantly coated (Figure 2 a2) while others were associated with only a few USPIO (Figure 2 a1). A schematic representation of TiONts/USPIO assemblies is shown on Figure 1c. Regarding iron oxide nanoparticles now, the mean size of USPIO measured by counting at least a hundred crystallites on HRTEM images, increased from (9.0 ± 2.0) nm before hydrothermal synthesis to (11.5 ± 2.0) nm after. This increase was probably due to Ostwald ripening in the basic NaOH solution necessary for TiONts formation. Characteristic distances of both TiONts and spinel phase were identified by SAED (Selected Area Electron Diffraction) of the specimen (Figure 3). All the rings can be indexed as those belonging to the two phases, and, no hematite formation was observed. Usually, the SAED pattern of TiONts presented another intense ring (halo rather than ring) corresponding to a d value of 0.76 nm corresponding to the interlayer distance between two consecutive tube walls. This distance was missing on this SAED pattern because it was hidden by the very close high intensity transmitted beam. On the other hand, all diffraction rings of maghemite were detectable except the low intensity ring of the (422)

plane (expected at 0.17 nm with a relative intensity of 8.3% with respect to the most intense plane).

b) a)

Zeta potential curves of TiONts and USPIO had the same profile: zeta potential was positive below the isoelectric point (respectively 3.6 and 7.0) and negative above (Figure 4). Consequently, there was a pH domain (3.6 -7.0) in which these two species can interact electrostatically. Hydrothermal synthesis of TiONts coated with USPIO was performed in NaOH (10 mol.L -1 , pH 14). After synthesis, the resulting product was dialysed until pH 5.5 was reached. TEM observations of this product showed that USPIO were grafted on TiONts (Figure 2 a1-a2). To exclude the possibility of an electrostatic binding, the previous sample was brought to pH 10 by NaOH addition under magnetic stirring. TiONts and USPIO assemblies remained the same (Figure 2 b1-b2). Consequently, the link between both oxide surfaces appeared not to be electrostatic. Preliminary washings have been realized first by a centrifugation step (25 000  g) followed by dialysis, both for uncoated nanotubes and for nanotubes/USPIO assemblies. Nanotubes formation is very sensitive to little variations in reaction parameters, in particular washing process, so first idea was to preserve the rolling-up mechanism leading to nanotubes by avoiding any change during washings. [START_REF] Kasuga | Titania nanotubes prepared by chemical processing[END_REF][START_REF] Papa | Effect of reaction parameters on composition and morphology of titanate nanomaterials[END_REF] This methodology lead to the assemblies presented in this paper. It is noteworthy that USPIO and TiONts are not separated during both intensive centrifugation and long dialysis time, nor by application of a magnetic field (Figure 1b). Finally, the cytotoxicity of these nanotubes was evaluated on CHO cells by MTT assay at 24 h.

The results showed that no cytotoxicity was observed up to 10.0 µg/mL (Figure 5 a).

The magnetic properties of these TiONts/USPIO assemblies were tested in vitro on a clinical 3 Tesla MRI. These nano-objects developed a magnetic response as we first observed by application of a magnetic field (Figure 1b). Indeed, the magnetic efficiency (r2 relaxivity) of this suspension, calculated with MRI phantoms (Figure 5 prove that USPIO coupled with TiONts do not lose their superparamagnetic property. With this r2 MRI measurement it is possible to estimate the density of USPIO coating. Briefly, the relaxivity of TiONts-USPIO is only due to the iron oxide nanoparticles and if the value of r2 of this composite material (0.59 L.mgnanoparticles -1 .s -1 ) is compared to the r2 of the naked USPIO used in this study (2.35 L.mgnanoparticles -1 .s -1 ) the weight ratio of USPIO on nanotubes may be obtained, here 25 %. The mean diameter of those maghemite nanoparticles is 11.5 nm and the density 5.2 (ICDD pattern 39-1346). So the mass of a single -Fe2O3 nanoparticle is 4.14 10 -18 g and 1 g of USPIO contains 2.4 10 17

nanoparticles. Since for 1 g of USPIO there are 3 g of TiONts, thanks to the surface area of the naked TiONts (121 m 2 .g -1 ) we found a density of coating of 6.7 10 -4 USPIO/nm 2 of TiONts. This value, linked to a weight ratio of 25 %, is much higher than the density of coating of the work of L. R. Hou et al. in the case of titanate coated by SnO2 24 and far higher than the one obtain by An et al. with their method of iron coating on TiO2 nanotubes [START_REF] An | Iron-coated TiO2 nanotubes and their photocatalytic performance[END_REF] . Our results are promising and are in any case sufficient to detect the nanotubes thanks to MRI.

For the first time, titanate nanotubes coating with USPIO are reported i) directly during hydrothermal synthesis of nanotubes and ii) without the use of an organic molecule linkage between both oxide particles. In comparison with a similar TiONts synthesis without USPIO, a distribution of nanotubes diameter was observed between 10 and 20 nm, which could be explained by the influence of USPIO on TiONts enrollment. TiONts coating was heterogeneous. After hydrothermal treatment in strongly basic conditions, iron oxide nanoparticles kept both their structure and their magnetic properties. A noteworthy increase of their mean crystallite size was nevertheless observed. By changing the pH and coupling with zeta potential and TEM observations, it appeared that interactions between nanotubes and USPIO were not electrostatic. Future studies will focus attention on the precise nature of these interactions (XPS and EXAFS experiments in progress). Moreover, these nanotubes have already been dispersed in water through different polymer graftings rendering the TiONts surface biocompatible. [START_REF] Millot | Dispersion of titanates nanotubes: comparison of PEI and PEG nanohybrids[END_REF] This new design of titanate needle shape nanocomposites, detectable by MRI, opens the door for future biodistribution studies of nanotubes to evaluate their potential as nanovectors and their toxicity.

EXPERIMENTAL SECTION USPIO synthesis. USPIO nanoparticles were prepared as described in the literature [START_REF] Belin | Structural variations as a function of surface adsorption in nanostructured particles[END_REF][START_REF] Maurizi | Easy Route to Functionalize Iron Oxide Nanoparticles via Long-Term Stable Thiol Groups[END_REF] ; the synthesis was optimized to obtain narrow grain size distribution. Briefly, a 1:2 molar ratio of ferrous and ferric chloride (respectively, 1 mol.L -1 and 2 mol.L -1 ) was added drop by drop to a 0.75 mol.L -1 NaOH solution at 90°C. After rinsing several times with HNO3 (1 mol.L -1 ) and water, the final suspension was maintained at pH 3 with an USPIO concentration of 44 mg.mL -1 .

TiONts coated USPIO synthesis. Titanate nanotubes were prepared by a classical hydrothermal method 2 . Fifty milligrams of titanium dioxide rutile precursor powder purchased from Tioxide were added to a NaOH aqueous solution (10 mol.L -1 , 110 mL). The same amount of USPIO was mixed with the dioxide suspension (1/1, w/w) i.e. 1136 L of the acidic suspension. pH of the suspension before hydrothermal treatment was 14. The mixture was submitted to ultrasound agitation (30 min, pulse 2 s, amplitude 50 %, 750 Watt, Sonics Vibra-Cells™) before being transferred to a sealed Teflon reactor under magnetic stirring (250 tr.min -1 ). The hydrothermal reactor temperature was kept at 150°C for 24 h. The resulting light brown colored product (Figure 1a) was isolated by centrifugation (25 000  g during 10 min) and washed with deionized water by dialysis until a pH 5.5 was attained (it approximately takes 3 days). Finally, the suspension was freeze-dried. Relaxation time measurements. Water proton transverse (T2) relaxation time measurements at 3.0 T were carried out at 298±1 K with a Siemens Magnetom Trio TIM using a commercially available birdcage head coil. Relaxation time measurements were performed on test tubes containing TiONts labelled with USPIO at different concentrations. For T2 determination, a multi-echo spin-echo (SE) pulse sequence was used, with TR = 5000 ms, matrix = 256x204, FOV = 100x80 mm and slice thickness = 5 mm. Images were acquired at 32 echoes, starting at TE = 7.9 ms with a 7.9 ms interval.

Image analysis was performed using ImageJ (Image Analysis Software developed by NIH, USA). The signal decay curve was fitted using a non-linear function to the equation S(TE) = A exp(-TE/T2). The relaxivity, r2, was then determined by fitting the relaxation rate (1/T2) nanoparticles concentration in mg.L -1 .

Figure 1 .

 1 Figure 1. Product of TiONts synthesis in presence of USPIO just after the hydrothermal treatment in NaOH 10 mol.L -1 and before dialysis: a) without and b) in the presence of a magnetic field. c)

Figure 2 .

 2 Figure 2. TEM images of TiONts coated with USPIO. These products were obtained (a1-a2) after

Figure 3 .

 3 Figure 3. a) SAED (150 cm camera length) of TiONts coated with USPIO and b) SAED profiles along

  Nevertheless, more intensive washings have been realized since, as centrifugation at various G (from 115  g to 25 000  g) alternate with ultrasounds but also ultrafiltration (with different membranes)

Figure 4 .

 4 Figure 4. Zeta potential curves of TiONts (dotted line) and USPIO (black line) as a function of pH (in NaCl 10 -2 mol.L -1 ). Isoelectric points of TiONts and USPIO were respectively about 3.6 and 7.0.

Figure 5 .

 5 Figure 5. a) MTT assay of TiONts on CHO cells at 24h. Cell viability is expressed as the percentage of

  b-c), gave an average value around 0.59 L.mgnanoparticles -1 .s -1 , indicating the possibility to detect USPIO decorated TiONts by MRI (Figure5 b). This value can be compare to the r2 values of naked TiONts and USPIO which are respectively 0 and 2.35 L.mgnanoparticles -1 .s -1 and

HRTEM

  Observations. HRTEM observations were performed on a JEOL JEM-2100 LaB 6 microscope operating at 200 kV and equipped with a high tilt pole piece achieving a point-to-point resolution of 0.25 nm. Zeta potential measurements. Zeta potentials were measured with a Malvern Nano ZS instrument supplied by DTS Nano V4.2 software.Cytotoxicity via MTT assay. Cytotoxicity was evaluated by measuring the mitochondrial dependent reduction of 3-(4,5-dimethyliazol-2-yl)2,5-diphenyltetrazolium bromide (MTT, Sigma) to formazan. Firstly, Chinese hamster ovary (CHO) cells (CHO-K1 cell line; ECACC) were cultured in DMEM media supplemented with 10% Fetal Bovine Serum before being stimulated in serum-deprived media when they reached 70% confluency. After 24 h of treatment, the cells were washed twice with PBS (NaCl 140 mMol.L -1 , KCl 2.7 mMol.L -1 , Na2HPO4 8.1 mMol.L -1 , and KH2PO4 1.5 mMol.L -1 ) and incubated with MTT (2 mL, 2 mg.mL -1 in Hanks medium) for 60 min at 37°C. Cells were then lysed by addition of the same volume of propane-2-ol and 0.1 mMol.L -1 HCl. The amount of formazan produced was detected by measurement of the absorbance at 570 nm with a spectrophotometer (VICTOR X3 apparatus, Perkin-Elmer).

ACKNOWLEDGMENTS

This work was supported by the Conseil Régional de Bourgogne. We would like to thank Dr. R.

Chassagnon for HRTEM images, J. Paris for his help during syntheses and Dr. Laure Dumont for the cytotoxicity tests.