Fatima Haidar

Annie Pradel

Yuan Chen
email: chen.yuan@hbut.edu.cn

Marie-Christine Record
email: m-c.record@univ-amu.fr

Deposition of Sb2Se3 Thin Films on Pt Substrate via Electro-Chemical Atomic Layer Epitaxy (EC-ALE)

Keywords: Underpotential deposition, EC-ALD, Cyclic voltammetry, Coulometry, AFM, Sb2Se3

This work deals with the deposition of a Sb2Se3 thin film on a Pt substrate using Electro-Chemical Atomic Layer Epitaxy (EC-ALE). We first investigated the UPD of selenium on a Pt substrate. Two routes have been evidenced to get a UPD layer of Se, either the polarization of the Pt-substrate at around 0.35 V in the Se solution or the deposition of full UPD Se together with bulk deposition of Se and dissolution of the latter one at lower potentials. The following investigation on the electrochemical behavior of antimony on selenium-covered platinum electrode by cyclic voltammetry and anodic potentiodynamic studies led us to choose the two-steps procedure for the Se deposition. After an optimization of the deposition parameters, the alternate deposition of Sb and Se elements to form the Sb2Se3 compound by EC-ALE on platinum substrate has been performed. The stoichiometry of the deposited film has been evaluated by coulometry and it has been confirmed by atomic force microscopy (AFM).

Introduction

Sb2Se3 is a V-VI semiconductor with a layered crystal structure (space group Pnma; n°62) [START_REF] Madelung | Semiconductors: other than group IV elements and III-V compounds[END_REF]. This compound is interesting as a thermoelectric material on two counts: first, it has a Seebeck coefficient of 46.2 μV/K in its polycrystalline form [START_REF] Rajpure | A comparative study of the properties of spraydeposited Sb2Se3 thin films prepared from aqueous and nonaqueous media[END_REF], and second, it is a component of the solid solutions (Bi,Sb)2(Te,Se)3, which are the best materials used to date for cooling applications [START_REF] Jeon | Electrical and thermoelectrical properties of undoped Bi2Te3-Sb2Te3 and Bi2Te3-Sb2Te3-Sb2Se3 single crystals[END_REF][START_REF] Yim | Compound tellurides and their alloys for Peltier cooling -A review[END_REF][START_REF] Yim | Thermoelectric properties of Bi2Te3-Sb2Te3-Sb2Se3 pseudo-ternary alloys in the temperature range 77 to 300[END_REF][START_REF] Rosi | Materials for thermoelectric generation[END_REF][START_REF] Xiao | Thermoelectrics Handbook: Macro to Nano[END_REF]. In addition, it also has promising applications in several other fields such as optoelectronic, photovoltaic and batteries. As a consequence, both elaboration and characterization of this compound have been widely studied (see for example Ref. [START_REF] Fernández | Preparation and characterization of Sb3Se3 thin films prepared by electrodeposition for photovoltaic applications[END_REF][START_REF] Rajpure | Preparation and characterization of spray deposited photoactive Sb2S3 and Sb2Se3 thin films using aqueous and non-aqueous media[END_REF][START_REF] Wang | Preparation and characterization of wire-like Sb2Se3 and flake-like Bi2Se3 nanocrystals[END_REF][START_REF] Xue | Pulsed laser deposited Sb2Se3 anode for lithium-ion batteries[END_REF][START_REF] Yu | Pulsed laser deposited heterogeneous mixture of Li2Se-Sb2Se3 nanocomposite as a new storage lithium material[END_REF][START_REF] Wang | Preparation and electrical transport properties of nanostructured Sb2Se3 films fabricated by combining spin-coating and gas-induced reduction[END_REF]). Miniaturization of the thermoelectric devices requires the use of thin films, which are usually prepared by physical methods under vacuum. Electrochemical deposition may provide an alternative process to these classical methods. Indeed, by contrast to other methods, electrochemical deposition is a low cost, room temperature production technique, which allows one to cover substrates with complex shapes. Some studies have already been reported on electrochemical deposition of Sb2Se3 [START_REF] Desai | Electrodeposition of Sb2Se3 thin films from alkaline bath[END_REF][START_REF] Torane | Preparation and characterization of electrodeposited Sb2Se3 thin films from non-aqueous media[END_REF][START_REF] Shi | Electrodeposition of Sb2Se3 on indium-doped tin oxides substrate: Nucleation and growth[END_REF][START_REF] Lai | Electrodeposition of antimony selenide thin films from aqueous acid solutions[END_REF]. Compared to other electrochemical methods, EC-ALE allows one to obtain deposits with flat surface which can easily be used for instance in the elaboration of superlattices. These nanostructures exhibit the highest Seebeck coefficients so far. The Electrochemical Atomic Layer Epitaxy (EC-ALE) technique, which was put forward by J. Stickney [START_REF] Gregory | Electrochemical atomic layer epitaxy (ECALE)[END_REF], was used in a previous work to deposit Sb2Se3 on a polycrystalline gold substrate [START_REF] Chen | Underpotential deposition of selenium and antimony on gold[END_REF]. This method is based on the alternate underpotential deposition (UPD) of atomic layers of the elements to make up a compound, advantageously combining the technique of electrochemical deposition and that of atomic layer epitaxy (ALE). Since separate solutions and potentials are used to deposit atomic layers of each element, all the steps of each cycle can be investigated and optimized independently. Ideally, one cycle of deposition results in the formation of one compound monolayer, the number of cycles determining the overall thickness of the complete electrochemical deposit. The process of electrodeposition is highly related to the nature of the substrate. However, in spite of the wide utilization of platinum as substrate material for electrodeposition, only few investigations involving this substrate have been carried out in the field of EC-ALE [START_REF] Yang | Formation and characterization of Sb2Te3 nanofilms on Pt by electrochemical atomic layer epitaxy[END_REF][START_REF] Zhu | Effect of potential on bismuth telluride thin film growth by electrochemical atomic layer epitaxy[END_REF][START_REF] Xiao | Electrodeposition and characterization of Bi2Se3 thin films by electrochemical atomic layer epitaxy (ECALE)[END_REF] and none of them concern the Sb2Se3 deposition. We recently investigated the deposition of Se and Sb on Pt substrates [START_REF] Chen | A voltammetric study on the atomic layer deposition of selenium and antimony on platinum electrode[END_REF]. It was shown that the Se-UPD layer with full coverage could be directly formed on a Pt substrate.

The cyclic voltammograms also showed that the two-step method needed for Se-UPD deposition on Au substrate [START_REF] Chen | Underpotential deposition of selenium and antimony on gold[END_REF], which consisted in the deposition of UPD together with bulk Se followed by the dissolution of bulk Se, could also be carried out on Pt substrate. The Sb underpotential deposition on Pt substrate is much more complex. It implies a multi-step process including an unavoidable preliminary adsorption of SbO + species on the Pt electrode, followed by their reduction to metallic Sb and further underpotential deposition of Sb on this Sb-modified Pt electrode to increase the coverage of the metallic Sb monolayer.

In this work, based on these previous results [START_REF] Chen | A voltammetric study on the atomic layer deposition of selenium and antimony on platinum electrode[END_REF], we investigated the electrochemical behavior of antimony on a selenium-covered platinum electrode by means of cyclic voltammetry, anodic potentiodynamic measurements and coulometry, and performed the EC-ALE process to elaborate Sb2Se3 thin films that we characterized by coulometry and atomic force microscopy (AFM).

Materials and methods

Reagents

Solutions were prepared using ACS reagent grade or higher-grade chemicals (Sigma-Aldrich, Inc., USA) and deionised water (Milli-Q > 18.2 MΩ•cm, Merck KGaA, Darmstadt, Germany). In this study three different solutions have been used: 1) the antimony solution containing Sb2O3 (99.999%) and the supporting electrolyte Na2SO4, with concentration of 0.05 mM and 0.2 M, respectively; 2) the selenium solution containing SeO2 (99.999%) and Na2SO4, with concentration of 0.5 mM and 0.2M, respectively; 3) the blank solution consisting in supporting electrolyte solution with same pH and ionic strength as the ones of deposition solutions. This latter one was used to rinse the electrochemical cell. The concentrations of Sb2O3 and SeO2 were chosen based on both literature data and experimental observations. The concentration used in literature for Sb2O3 solutions [START_REF] Yang | Formation and characterization of Sb2Te3 nanofilms on Pt by electrochemical atomic layer epitaxy[END_REF][START_REF] Venkatasamy | ALD approach toward electrodeposition of Sb2Te3 for phase-change memory applications[END_REF][START_REF] Yang | Electrochemical aspects of depositing Sb2Te3 compound on Au substrate by ECALE[END_REF][START_REF] Gao | Deposition of antimony telluride thin film by ECALE[END_REF][START_REF] Zhu | Development of growth cycle for antimony telluride film on Au (111) disk by electrochemical atomic layer epitaxy[END_REF][START_REF] Wade | Electrochemical atomic-layer epitaxy: electrodeposition of III-V and II-VI compound semiconductors[END_REF][START_REF] Wade | Electrochemical formation of a III-V compound semiconductor superlattice: InAs /InSb[END_REF][START_REF] Zhu | Electrochemical aspects and structure characterization of VA-VIA compound semiconductor Bi2Te3/Sb2Te3 superlattice thin films via Electrochemical Atomic Layer Epitaxy[END_REF] ranges from 0.01 mM to 0.05mM, and we can confirm from experiment that 0.05 mM which is the most often used concentration, is higher than the solubility limit of antimony oxide in sulfuric acid solution. The concentration used in literature for SeO2 solutions [START_REF] Xiao | Electrodeposition and characterization of Bi2Se3 thin films by electrochemical atomic layer epitaxy (ECALE)[END_REF][START_REF] Lister | Formation of the first monolayer of CdSe on Au(111) by electrochemical ALE[END_REF][START_REF] Lister | Electrochemical formation of CdSe monolayers on the low index planes of Au[END_REF][START_REF] Mathe | Deposition of CdSe by EC-ALE[END_REF][START_REF] Loglio | Cadmium selenide electrodeposited by ECALE: electrochemical characterization and preliminary results by EXAFS[END_REF][START_REF] Pezzatini | Formation of ZnSe on Ag(111) by electrochemical atomic layer epitaxy[END_REF][START_REF] Zhang | Optimization of electrochemical aspects for epitaxial depositing nanoscale ZnSe thin films[END_REF][START_REF] Mathe | Formation of HgSe thin films using Electrochemical Atomic Layer Epitaxy[END_REF][START_REF] Venkatasamy | Optimization studies of HgSe thin film deposition by electrochemical atomic layer epitaxy (EC-ALE)[END_REF][START_REF] Vaidyanathan | Quantum confinement in PbSe thin films electrodeposited by electrochemical atomic layer epitaxy (EC-ALE)[END_REF][START_REF] Qiao | Fabrication of Sn-Se compounds on a gold electrode by electrochemical atomic layer epitaxy[END_REF][START_REF] Vaidyanathan | Formation of In2Se3 thin films and nanostructures using electrochemical atomic layer epitaxy[END_REF][START_REF] Vaidyanathan | Electrodeposition of Cu2Se thin films by electrochemical atomic layer epitaxy (EC-ALE)[END_REF][START_REF] Vaidyanathan | Preliminary studies in the electrodeposition of PbSe/PbTe superlattice thin films via Electrochemical Atomic Layer Deposition (ALD)[END_REF] ranges from 0.05 mM to 2 mM, with 0.5 mM as the most often used one. The pH values for all the solutions were adjusted to 1.00 with H2SO4. All the solutions were freshly prepared and degassed for 30 minutes with pure Ar gas (Ar Bip, O2 < 10 ppb, Air Products and Chemicals, Inc., France) just before starting each series of measurements. All the solutions were maintained under Ar atmosphere during the experiments, which were performed at room temperature.

Substrates

The platinum substrates consisted of quartz slides, coated with 200 nm thick platinum films (99.9% pure) by physical vapor deposition. A thin layer of Cr (25 nm) was beforehand deposited under the Pt film to improve the adhesion of platinum onto quartz. Before use, the platinum substrates were first annealed at 375 °C for 18 h in sealed glass tubes under vacuum (10 -6 Torr) and then soaked in hot nitric acid for 5 min. Before each experiment, in order to clean and check the substrate, cyclic voltammetry (CV) scanning (repeated 25 times) of the Pt substrate in 0.1 M H2SO4 solution was performed with potential varying from -0.27 to 1.20 V. The real area of the Pt electrode was estimated to be 3.79 cm 2 . This estimation was done from the I-t curve of CV by integrating the reduction peaks of adsorbed hydrogen ions (see our previous paper [START_REF] Chen | A voltammetric study on the atomic layer deposition of selenium and antimony on platinum electrode[END_REF]).

Electrochemical equipment

All the electrochemical experiments were performed with the EC-ALE equipment developed in our group and described in our previous works [START_REF] Chen | Underpotential deposition of selenium and antimony on gold[END_REF][START_REF] Chen | A voltammetric study of the underpotential deposition of cobalt and antimony on gold[END_REF]. In the electrochemical cell, a platinum sheet (Sigma-Aldrich, Inc., USA) is used as counter electrode. The working electrode is made of platinum substrate. All electrochemical potential values are measured with respect to the potential of an Ag/AgCl reference electrode (3.0 M NaCl, 0.209 V vs. standard hydrogen electrode (SHE), Ametek, Inc., USA). The atomic layer deposition of Se and Sb elements was electrochemically studied by means of cyclic voltammetry, anodic potentiodynamic scanning and coulometry.

Deposit characterization

The Sb2Se3 thin films were characterized by coulometry and atomic force microscopy (AFM). The AFM measurements were performed with a Bruker Nanoscope Dimension 3100 atomic force microscope (AFM, Bruker, Palaiseau, France) in the tapping mode under ambient conditions. The cantilever tips were Silicon Point Probe Plus NCSTR (Nanosensors, Neuchâtel, Switzerland; force constant 6.5 N/m, resonance frequency 157 kHz). All the image treatments were performed using Gwyddion [45].

Results and discussion

As noted in the Introduction section, the formation of both Sb and Se atomic layers on Pt was already investigated in our previous paper [START_REF] Chen | A voltammetric study on the atomic layer deposition of selenium and antimony on platinum electrode[END_REF]. The results are as follows.

As it was shown in literature [START_REF] Wu | An EQCM study of Sb adsorption and coadsorption with CO on Pt electrode in perchloric acid solutions[END_REF][START_REF] Herrero | Poison formation reaction from formic acid on Pt(100) electrodes modified by irreversibly adsorbed bismuth and antimony[END_REF][START_REF] Fernandez-Vega | Heterogeneous electrocatalysis on well defined platinum surfaces modified by controlled amounts of irreversibly adsorbed adatoms: Part II. Formic acid oxidation on the Pt (100)-Sb system[END_REF][START_REF] Feliu | New observations of a structure sensitive electrochemical behaviour of irreversibly adsorbed arsenic and antimony from acidic solutions on Pt (111) and Pt (100) orientations[END_REF][START_REF] Kizhakevariam | Structure and reactivity of bimetallic electrochemical interfaces: infrared spectroscopic studies of carbon monoxide adsorption and formic acid electrooxidation on antimony-modified Pt(100) and Pt(111)[END_REF][START_REF] Sun | Electrocatalytic properties of glassy metals: Combined Auger electron spectroscopy and electrochemical studies of the surface composition and electrocatalysis of CO oxidation at amorphous Pt66Sb34 alloy[END_REF][START_REF] Sun | Electrocatalytic Oxidation of Formic Acid and Methanol at the Amorphous Pt66Sb34 Electrode[END_REF] that antimony can adsorb irreversibly on Pt surface by simply immersing Pt electrode in a solution containing SbO + ions at the OCV, we designed the following experimental procedure to adsorb adequate Sb species on a clean Pt electrode : (i) Before the beginning of the experiments, the clean Pt electrode is placed in the electrochemical cell filled with blank solution ;

(ii) the Sb solution is introduced into the electrochemical cell and kept flowing for 5 s. Then the Sb solution is kept still for 15 s ; (iii) the step (ii) as a cycle is repeated totally for 40 times, and the variation of the OCV of Pt electrode during this process is recorded (see red curve in Figure 1) ; (iv) as a control, a similar experiment procedure is performed as steps (i) to (iii), but during the whole process the blank solution is used instead of the Sb solution. The evolution of the OCV of Pt electrode during this process is shown as the black curve in Figure 1.

From the results shown in Figure 1

UPD of Selenium on Pt substrate (Pt/Se)

The underpotential deposition of Se element on Pt substrate was carried out on the basis of our previous investigation reported in [START_REF] Chen | A voltammetric study on the atomic layer deposition of selenium and antimony on platinum electrode[END_REF]. At the time, it was shown that the underpotential deposition of Se occurred at 0.48 V (with respect to the potential of Ag/AgCl reference electrode) and its bulk deposition occurred at 0.24 V, which leads us to conclude by comparison with results of ref. [START_REF] Iqbal | Quantitative Determination of H2Se at Model Metal fcc(111) Selenide Surface: DEMS, STM, and AFM Studies[END_REF], that Se is preferentially deposited onto Pt(111). As with the UPD of Se on Au substrate [START_REF] Chen | Underpotential deposition of selenium and antimony on gold[END_REF], two additional reductions were evidenced at lower potentials: 1) the reduction of 3-D bulk-deposition of Se to H2Se, which is soluble, at -0.34V, 2) the formation of red Se due to the coexistence in the cell of HSeO 3-and H2Se at -0.4V. Therefore there are two possibilities to get a UPD layer of Se, either the polarization of the Pt-substrate at around 0.35 V in the Se solution or the deposition of full UPD Se together with bulk deposition of Se and dissolution of the latter one at lower potentials. For the first Se layer, both procedures are relevant.

However, a polarization at 0.35 V cannot be used for the deposit in the subsequent cycles since the stripping of UPD-Sb on Se-covered Pt substrate starts occurring below 0.35V, as demonstrated below in section 2.2. Therefore, the two-steps procedure was used in the present work. The potential window to deposit both UPD and bulk Se is [-0.25V, 0.1V] as evidenced in [START_REF] Chen | A voltammetric study on the atomic layer deposition of selenium and antimony on platinum electrode[END_REF]. Thus, the procedure for the first layer deposition will be as follows: i) the Pt electrode is rinsed for 15 s in the Se solution at OCV and kept for 10 s in this solution at 0 V to deposit the bulk Se ; ii) while the electrode is maintained at the same potential the cell is rinsed with blank solution for 10 s to get rid of HSeO3 -ions in order to avoid the formation of red Se ; iii) the potential is moved down to -0.36 V and maintained at this value for 10 s while the blank solution is rinsing such that bulk Se is reduced to HSe -.

UPD of antimony on Se-covered Pt substrate (Pt/Se/Sb)

The investigation of the UPD of antimony on Se-covered Pt was performed as follows: (i) the previously prepared UPD Se-covered electrode was kept at 0.45 V, a potential where the UPD-Se layer is stable, while the Sb solution was introduced into the cell; (ii) the UPD Se-covered electrode was scanned from 0.45 V to -0.35 V and positively back to 1.05 V.

Figure 2 shows the CV curve of the Se-covered Pt electrode in Sb solution. C1 and C2 peaks are assigned to the UPD peaks of Sb on Se-covered Pt electrode and A2, A3 and A4 their corresponding stripping peaks. Such assignment is based on a detailed study of the peak evolution during anodic potentiodynamic scanning after different polarizations reported in the following part of the present section. In agreement with previous results [START_REF] Gregory | Electrochemical atomic layer epitaxy (ECALE)[END_REF], the additional peaks appearing in the anodic part of the curve in the range [0.7V -1V] correspond to the stripping peaks of Se UPD on the Pt electrode. No peak that could be assigned to bulk Sb deposition or stripping, is observed in the CV curve. It could be due to the fact that bulk Sb deposition, which is closely related to the concentration of the solution (0.05 mM), is slow and cannot occur to a large extent during a relatively fast cyclic voltammogram (20 mV/s). This assumption is supported by the results presented just below that show the need for long polarization duration to observe the appearance of the stripping peak of bulk Sb. One can find similar electrochemical behavior of antimony in the literature [START_REF] Gao | Deposition of antimony telluride thin film by ECALE[END_REF][START_REF] Zhu | Development of growth cycle for antimony telluride film on Au (111) disk by electrochemical atomic layer epitaxy[END_REF][START_REF] Yang | Electrochemical aspects of depositing Sb2Te3 compound on Au substrate by ECALE[END_REF][START_REF] Yang | Formation and characterization of Sb2Te3 nanofilms on Pt by Electrochemical Atomic Layer Epitaxy[END_REF]. In order to determine the optimum potential for Sb UPD onto Se-covered Pt, i.e. the potential leading to a high coverage of Sb UPD while avoiding the Sb bulk deposition, we performed an anodic potentiodynamic scanning experiment. The Se-covered Pt electrode was polarized for 20 s at various potentials (from -0.16 V to -0.26 V) while the Sb solution was kept flowing through the cell to provide enough Sb ions. Then the cell was rinsed by the blank solution and the anodic potential scanning was performed up to 1.05 V. The current curves are given in Fig. 3a. When the polarization potential was equal or higher than -0.20 V, five peaks (A2 -A6) only were observed on the curve.

For potentials equal or lower than -0.21 V an additional peak, A1, could be seen and its intensity increased as the polarization potentials decreased. Therefore, the peak A1 should correspond to the oxidative stripping peak of the bulk deposited Sb. According to these results, a potential higher than -0.20 V has to be chosen for the UPD Sb on Se-covered Pt substrate.

Since the Sb concentration of the solution is low (0.05 mM), the Sb bulk deposition should be slow.

However, to ensure that bulk deposition of Sb was avoided, we performed two additional experiments.

The Se-covered Pt electrode was polarized for different durations at -0.20 V (Fig. 3b) and -0.19 V (Fig. 3c) in a flowing Sb solution to provide enough Sb ions, then the cell was rinsed with blank solution and an anodic potential scanning was performed up to 1.05 V with a scanning rate of 20 mV/s. Figure 3. Anodic potentiodynamic scanning curves of the Se-covered Pt substrate obtained in the blank solution at pH 1.0: i) after polarization for 20 s at various potentials (Fig. 3a); ii) after polarization for different durations, at -0.20 V (Fig. 3b), at -0.19 V (Fig. 3c). In all cases, the polarizations were performed in a flowing Sb solution and the cell was rinsed before the anodic potentiodynamic measurements. The scanning rate was 20 mV / s.

As shown in Fig. 3b, the A1 peak corresponding to the oxidation of the bulk Sb deposition is observed when the polarization potential is -0.20 V for a time duration of 40 s and 60 s. By contrast, when the polarization potential is -0.19 V, irrespective of the time duration, this peak does not appear (see Fig. 3c).

Therefore, the ideal potential for Sb underpotential deposition on a Se-covered Pt electrode seems to be -0.19 V and the chosen procedure is as follows. After having rinsed the cell for 60 s with the Sb solution, the Pt electrode is kept in this solution for 10 s at -0.19 V and then rinsed with blank solution at the same potential for 15 s.

Elaboration and characterization of Se-Sb compound for three EC-ALE cycles (6 atomic layers)

Alternative deposition of Se and Sb

The underpotential deposition parameters for the first three EC-ALE cycles were defined based on the above results. They are given in Table 1.

The potential window to deposit both UPD and bulk Se is [-0.25V, 0.1V] as evidenced in [START_REF] Gregory | Electrochemical atomic layer epitaxy (ECALE)[END_REF]. Thus, during the second and third cycles, in order to narrow the potential variations in the system, a potential similar to the value used for Sb UPD was used for the Se deposition.

Owing to changes in the atom environment and in the film resistance during the completion of the successive depositions, the deposition parameters had to be adjusted and are thus somewhat different from cycle to cycle.

Generally, as the alternative deposition progresses, the deposited part will generate additional resistance [START_REF] Gao | Deposition of antimony telluride thin film by ECALE[END_REF][START_REF] Zhu | Development of growth cycle for antimony telluride film on Au (111) disk by electrochemical atomic layer epitaxy[END_REF][START_REF] Yang | Electrochemical aspects of depositing Sb2Te3 compound on Au substrate by ECALE[END_REF][START_REF] Yang | Formation and characterization of Sb2Te3 nanofilms on Pt by Electrochemical Atomic Layer Epitaxy[END_REF], which will cause the deposition potential to shift negatively. In our experiments, we tried different negatively-shifted potentials for deposition, and then determined the deposition potential of each layer according to the amount of stripping from anodic potentiodynamic scanning curve. The optimization of the experimental parameters has to be done at the beginning of the deposition. After three cycles, the latter values can be used for all the subsequent cycles, which is why we limited the deposition to 3 cycles.

For each cycle in step 1, the solution containing the reactant (I for Se, II for Sb) is introduced into the cell and is kept flowing in it during few seconds to properly fill in the cell. Step 2 corresponds to the deposition one ; the solution is then maintained static to avoid perturbations in the process. In step 3, the cell is rinsed with the blank solution flowing in it for few seconds. In step 4, when relevant, the applied potential is greatly reduced to dissolve bulk Se. After each cycle, an anodic potentiodynamic scanning was performed. The corresponding curves are plotted in Fig. 4. In all cases, the stripping peaks of both Sb and Se can be observed. For the first layer the curve is in agreement with those presented in Figure 3. There are 3 stripping peaks at around 0.1 V, 0.45 V and 0.6 V for Sb and 2 stripping peaks at around 0.75 V and 0.9 V for Se. For the subsequent layers, additional peaks appear around 0.6V, they can be related to the stripping of Se atoms deposited onto Sb ones.

Coulometry analysis

After deposition, each atomic layer is immediately removed using the blank solution, and the amount of electric charges is calculated by integrating the corresponding oxidative peak in current-time curve while deducting the background current. The results are shown in Fig. 5. As expected, the calculated charges for both Se and Sb deposits increase linearly with the number of cycles. The corresponding slopes are 981 μC cm -2 and 482 μC cm -2 , respectively. The coverage rate for both Se and Sb can be estimated from these results ; the values are 0.7 ML and 0.5 ML, respectively. Since the reduction of Se and Sb involves four and three electrons, respectively, the determination of the stripping charges leads to a stoichiometric ratio of 3.06:2 for Se to Sb. This stoichiometric ratio approximates that of the Sb2Se3 compound.

Atomic Force Microscopy

The morphology of the deposited layer analyzed by AFM is shown in Figure 6a Figure 7 shows the shift in frequency of the cantilever resonance as a function of the bias voltage applied to the tip to null out the contact potential for the Pt/tip system (Fig. 7a) and for the Sb2Se3-Pt/tip system (Fig. 7b). In each case, the data are well fitted with a quadratic function; the corresponding equations are given in the figure. This evolution agrees with the quadratic variation of the electrostatic force with the applied bias voltage [START_REF] Sweetman | AFM of self-organised nanoparticle arrays: frequency modulation, amplitude modulation, and force spectroscopy in Nanostructured Thin Films[END_REF]. The maximum of the frequency shift corresponds to a voltage of -23,5 mV for the Pt/tip system and of 255 mV for the Sb2Se3-Pt/tip one. The bias voltage is much higher for the Sb2Se3-

  , one can see that the OCV of Pt electrode decreases continuously in the Sb solution, indicating that Sb species (SbO + ) are indeed adsorbed irreversibly on Pt electrode. Before the beginning of the experiments, the clean Pt electrode is kept in the electrochemical cell filled with blank solution, where the OCV is approximately 0.70 V. The high OCV is due to the chemical oxygen Ochem adsorbed on Pt electrode in an acid solution environment according to the literature [46]. When the Sb solution is introduced into the cell, the SbO + species are irreversibly adsorbed on Pt and replace the adsorbed chemical oxygen Ochem at their sites, leading to the structural change of the double-layer on the surface of Pt electrode. The structural change of the double-layer leads to the change of OCV. Therefore, the decrease of the OCV demonstrates the occurrence of the irreversible adsorption of Sb species. The OCV of Pt electrode in Sb solution approximately reaches a plateau after 800 s (40 cycles), indicating that the amount of irreversibly adsorbed Sb species reaches a maximum. This observation also illustrates that the irreversible adsorption process of Sb species on Pt substrate is kinetically slow, or in other words, the process of replacing Ochem by SbO + species is kinetically slow. At the opposite, the underpotential deposition of Se on Pt substrate was quite straightforward. Therefore, in the present work, we started the alternate deposition of Sb and Se elements by first depositing Se atomic monolayer onto the Pt electrode.

Figure 1 .

 1 Figure 1. Evolution of the OCV of Pt electrode during the irreversible adsorption process of Sb (red curve) compared to a reference experiment with blank solution (black curve).

Figure 2 .

 2 Figure 2. Voltammograms of Se-covered Pt substrate in the Sb solution at pH 1.0. The scanning rate is 20 mV/s.

Figure 4 .

 4 Figure 4. Anodic potentiodynamic scanning curves obtained after each cycle (Se + Sb)

Figure 5 .

 5 Figure 5. Charges involved in the oxidative stripping of Se and Sb as a function of the number of EC-ALE cycles.

Figure 6 .

 6 Figure 6. Atomic force microscopy (AFM) micrographs of (a) 3 cycle-Sb-Se deposit on Pt substrate and (b) bare Pt substrate.

Figure 7 .

 7 Figure 7. Frequency shift vs. bias voltage with the tip placed above (a) the Sb2Se3-Pt system, (b) the Pt substrate. The data are fitted with a quadratic function.

Table 1 .

 1 Electrodeposition parameters of the first three EC-ALE cycles for Sb-Se compound , II, and III represent the flowing Se, Sb, and blank solutions, respectively. I s and II s represent the static Se and Sb solution, respectively. OCV: open circuit voltage

	Cycles Elements Step 1	Step 2	Step 3	Step 4
	Cycle 1 Se	I, OCV, 15 s	I s , 0 V, 15 s	III, 0 V, 10 s	III, -0.36 V, 10 s
	Sb	II, -0.19 V, 60 s II s , -0.19 V, 10 s III, -0.19V, 15 s
	Cycle 2 Se	I, -0.19 V, 15 s	I s , -0.19 V, 15 s III, -0.19 V, 10 s III, -0.40 V, 10 s
	Sb	II, -0.20 V, 45 s II s , -0.20 V, 25 s III, -0.20 V, 15 s
	Cycle 3 Se	I, -0.20 V, 15 s	I s , -0.20 V, 15 s III, -0.20 V, 10 s III, -0.40 V, 10 s

I


Rebecca Deprez-Poulain
email: rebecca.deprez@univ-lille2.fr

Marion Flipo

Catherine Piveteau

Florence Leroux

Sandrine Dassonneville

Isabelle Florent

Louis Maes

Paul Cos

Benoit Deprez
email: benoit.deprez@univ-lille2.fr

Structure-activity relationships and blood distribution of antiplasmodial aminopeptidase-1 inhibitors

Keywords: proteases, metalloenzymes, drug targets, protease inhibitors, plasmodium, antimalarial agents AcOEt, ethyl acetate, AUC, area-under-curve, BTFA, boron tris(trifluoroacetate), APN : Neutral Aminopeptidase, CA, carbonic anhydrase, CH3CN, acetonitrile, DCM, dichloromethane

Malaria is severe infectious disease that causes between 655,000 and 1.2 million deaths annually. To overcome the resistance to current drugs, new biological targets are needed for drug development. Aminopeptidase M1 (PfAM1), a zinc metalloprotease, has been proposed

as a new drug target to fight malaria. Herein, we disclosed the structure-activity relationships of a selective family of hydroxamate PfAM1 inhibitors based on the malonic template. In particular, we performed a "fluoro-scanning" around hit 1 that enlightened the key positions of the halogen for activity. The docking of the best inhibitor 2 is consistent with in vitro results. The stability of 2 was evaluated in microsomes, plasma and towards glutathione. The in vivo distribution study performed with the nanomolar hydroxamate inhibitor 2(BDM14471) revealed that it reaches its site of action. However, it fails to kill the parasite at concentrations relevant to the enzymatic inhibitory potency, suggesting that killing the parasite remains a challenge for potent and drug-like catalytic-site binding PfAM1 inhibitors. In all, this study provides important insights for the design of inhibitors of PfAM1, and the validity of this target.

INTRODUCTION

The WHO/TDR initiative promotes drug target validation for neglected diseases 1 . As such, innovative strategies are being developed to discover new antiplasmodial targets and may include a target-driven approach by cell-based screening 2 , drug repositioning and chemogenomics 3,4 . In this context, the Plasmodium proteases are putative targets [5][6][START_REF]N-Benzyl-2-(4-fluoro-benzyl)-N'-hydroxy-malonamide (8). White powder; Yield 23%; Purity 95%[END_REF][START_REF]OH); 10.47 (s; CONHO); tR LCMS 4.20 min[END_REF] . Their study benefits from the successful work done on proteases in other pathologies, such as AIDS, hypertension and type-II diabetes 9, 10 . Among the metalloproteases of Plasmodium, falcilysin is involved in the intravacuolar digestion of globin and has no reported inhibitor 11 . On the other hand, aminopeptidases degrading oligopeptides resulting from haemoglobin digestion are well-studied [12][START_REF] Gardiner | Plasmodium falciparum: new molecular targets with potential for antimalarial drug development[END_REF][START_REF] Gavigan | The role of aminopeptidases in haemoglobin degradation in Plasmodium falciparum-infected erythrocytes[END_REF] . Recently, inhibitors of LAP (Leucine-aminopeptidase), a hexameric aminopeptidase of the M17-family, have been described [START_REF] Gardiner | Overexpression of leucyl aminopeptidase in Plasmodium falciparum parasites. Target for the antimalarial activity of bestatin[END_REF][START_REF] Skinner-Adams | Identification of Phosphinate Dipeptide Analog Inhibitors Directed against the Plasmodium falciparum M17 Leucine Aminopeptidase as Lead Antimalarial Compounds[END_REF] .

Florent et al characterized the aminopeptidase M1 (PfAM1 or PfM1AAP) that shows a strict aminopeptidase activity at pH 7.4 and a broad substrate spectrum [START_REF] Allary | Properties, stage-dependent expression and localization of Plasmodium falciparum M1 family zinc-aminopeptidase[END_REF][START_REF] Florent | A Plasmodium falciparum aminopeptidase gene belonging to the M1 family of zinc-metallopeptidases is expressed in erythrocytic stages[END_REF] . PfAM1 localizes differently in trophozoites and schizonts, suggesting a particular role in the intraerythrocytic life of the parasite. PfAM1 degrades oligopeptides resulting from haemoglobin digestion either in the vacuole [START_REF] Ragheb | Distribution and Biochemical Properties of an M1-family Aminopeptidase in Plasmodium falciparum Indicate a Role in Vacuolar Hemoglobin Catabolism[END_REF] or the cytosol [START_REF] Klemba | A Plasmodium falciparum dipeptidyl aminopeptidase I participates in vacuolar hemoglobin degradation[END_REF] . The inhibition of PfAM1 by bestatin analogues induces swollen digestive vacuoles due to the accumulation of oligopeptides [START_REF] Harbut | Bestatin-based chemical biology strategy reveals distinct roles for malaria M1-and M17-family aminopeptidases[END_REF] . Also, a role in the reinvasion of erythrocytes has been found, in agreement with the observation of Kitjaroenthan et al [START_REF] Kitjaroentham | Effect of metalloprotease inhibitors on invasion of red blood cell by Plasmodium falciparum[END_REF] . Studies on PfAM1 localization suggest that it is trafficked via the parasitophorous vacuole [START_REF] Azimzadeh | Plasmodium falciparum PfA-M1 aminopeptidase is trafficked via the parasitophorous vacuole and marginally delivered to the food vacuole[END_REF] . PfAM1 is also present in the nucleus, but so far no function can be related to its nuclear localization [START_REF] Ragheb | Distribution and Biochemical Properties of an M1-family Aminopeptidase in Plasmodium falciparum Indicate a Role in Vacuolar Hemoglobin Catabolism[END_REF] .

Given these results, PfAM1 can be considered an attractive candidate to initiate screening and lead optimization. Our research group was the first to describe PfAM1 screening and the first two series of hydroxamic non-peptidic non-selective PfAM1 inhibitors with a mixed mode of action [START_REF] Flipo | synthesis and antimalarial activity of novel, quinoline-Based, zinc metallo-aminopeptidase inhibitors[END_REF] . The screening of a focused library of zinc-binding molecules introduction of steric constrains at the malonic position and led to the discovery of 2, a nanomolar selective inhibitor of PfAM1. 2 shows good physico-chemical, in vitro plasma stability and promising antiplasmodial activity in vitro [START_REF] Flipo | Novel Selective Inhibitors of the Zinc Plasmodial Aminopeptidase PfA-M1 as Potential Antimalarial Agents[END_REF] . Other inhibitors, such as phosphonopeptides, were subsequently published [START_REF] Cunningham | Chemical Target Validation Studies of Aminopeptidase in Malaria Parasites Using {alpha}-Aminoalkylphosphonate and Phosphonopeptide Inhibitors[END_REF][START_REF] Velmourougane | Synthesis of New (-)-Bestatin-Based Inhibitor Libraries Reveals a Novel Binding Mode in the S1 Pocket of the Essential Malaria M1 Metalloaminopeptidase[END_REF] and were designed as mixed inhibitors of the aminopeptidases of the M1 and M17 families. The binding of one non-selective compound to PfAM1 was assessed by co-crystallisation with the recombinant enzyme whose X-ray structure had been published [START_REF] Mcgowan | Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase[END_REF] . Recently, peptide-based bestatin analogues were also disclosed [START_REF] Velmourougane | Synthesis of New (-)-Bestatin-Based Inhibitor Libraries Reveals a Novel Binding Mode in the S1 Pocket of the Essential Malaria M1 Metalloaminopeptidase[END_REF] .

This paper reports the further optimization of our inhibitors whereby initial medicinal chemistry experiments revealed that the introduction of a fluorine on the scaffold had a significant impact on the inhibitory activity [START_REF] Flipo | Novel Selective Inhibitors of the Zinc Plasmodial Aminopeptidase PfA-M1 as Potential Antimalarial Agents[END_REF] . First, the effect on potency of systematic moving of fluorine ("fluorine scanning") [START_REF] Morgenthaler | A fluorine scan of non-peptidic inhibitors of neprilysin: Fluorophobic and fluorophilic regions in an enzyme active site[END_REF][START_REF] Huang | Exhaustive Fluorine Scanning toward Potent p53-Mdm2 Antagonists[END_REF] around compounds 1 and 2 was evaluated. Also substitution at the malonic carbon and the introduction of a pyridine to introduce a positive charge, which is a hallmark of many antiplasmodials, were explored. Analogues on PfAM1 covering a 2.5 log range of IC50s were tested in vitro on the intraerythrocytic cycle. We analyze these results in the light of structure-activity relationships on the isolated enzyme and distribution/stability data of the most active PfAM1 inhibitor.

Synthesis of inhibitors 1-14

Inhibitors were synthesized as described in schemes 1 and 2. Precursors 5a-b,d-e and 1-4,12 were synthesized as previously described [START_REF] Flipo | Novel Selective Inhibitors of the Zinc Plasmodial Aminopeptidase PfA-M1 as Potential Antimalarial Agents[END_REF][START_REF] Flipo | Hydroxamates: Relationships between Structure and Plasma Stability[END_REF] . 5c was synthesized by a Knoevenagel reaction between 4-fluorobenzaldehyde and ethyl N-tert-butoxymalonamate in refluxed ethanol in the presence of piperidine. The resulting ester was saponified to give carboxylic acid 5c as a single E-isomer as determined by ROESY NMR. 5a-d reacted with the corresponding amine after activation of the carboxylic acid function using either ethylchloroformate or EDCI/HOBt. The deprotection of the hydroxamate was performed in acidic conditions to give compounds 6-11,13-14.

O O O N H O R O O H O N H O N H O O H O O F O O O O O O N H O Trt O H O O O O H O O O O N H O O H O O F R = -Bzl R = -H R= -Bzl R=H (E)
Compound 12 was obtained from 5e by coupling first the 4-fluorobenzylamine. Then, the ester was saponified, and the resulting carboxylic acid was activated and reacted with trityleprotected hydroxylamine. The final deprotection was performed in TFA/DCM.

O N H O O H R OR 3 O N H O N H R 1 OH R O O O O H N H R 1 N H OH O O 1)

PfAM1 inhibition and selectivity towards pAPN.

In these series, hit 1 (IC50 = 27 nM) and its analogue 2 (IC50 = 6 nM) bearing a pfluorobenzene ring were more active than 4 (IC50 =125 nM) deprived of a halogen (Table 1). [START_REF] Flipo | Novel Selective Inhibitors of the Zinc Plasmodial Aminopeptidase PfA-M1 as Potential Antimalarial Agents[END_REF] Fluorine is often used in medicinal chemistry [START_REF] O'hagan | Fluorine in health care: Organofluorine containing blockbuster drugs[END_REF][START_REF] Gakh | Extreme modulation properties of aromatic fluorine[END_REF][START_REF] Smart | Fluorine substituent effects (on bioactivity)[END_REF] as it is sterically small and most often chemically inert. It is the most electronegative halogen and forms stable bonds with carbon which are poorly polarizable. Compounds 6-8, resulting from "fluorine scanning", display a ten-fold decrease in potency. This illustrates the importance of the presence of fluorine in the para-position of the benzylamido moiety rather than in ortho-or meta-position or on the other phenyl ring. Interestingly, compound 9 that has a fluorine atom at this critical position, and an additional fluorine, is less active than compound 1. This finding suggests that fluorine on the benzyl malonyl moiety is exposed to a fluorophobic region. The same trend is observed for unsaturated compounds 10 and 11, analogues of 2 (IC50 = 6 nM), that are seven-to ten-fold less potent. Nevertheless, the beneficial unsaturation compensates for bad fluorine position.

Substitution of a hydrogen atom by a methyl group on the malonic carbon of 1 (compound 12) induced a two-fold loss in activity, while the substitution of para-fluorophenyl by a pyridine ring [START_REF] Saleh | Synthesis and antimicrobial activity of 2-fluorophenyl-4,6-disubstituted [1,3,5]triazines[END_REF] in compounds 1 and 2 enabled the exploration of the properties of the enzyme pocket and generated a positive charge. Compounds 13 and 14 were about 50 times less active than their analogue.

All compounds were shown to be selective for the parasitic enzyme versus the mammalian enzyme APN (neutral aminopeptidase). The highest selectivity ratios were observed for compounds 2 and 11. To rationalize structure-activity relationships, compound 2 was docked in PfAM1 (using enzyme in PDB: 3T8V) (Figure 1). The hydroxamate moiety binds the Zn 2+ ion and is involved in a hydrogen-bond with Glu497. The S1 pocket of the enzyme is hydrophobic and able to undergo major conformational changes upon ligand binding [START_REF] Velmourougane | Synthesis of New (-)-Bestatin-Based Inhibitor Libraries Reveals a Novel Binding Mode in the S1 Pocket of the Essential Malaria M1 Metalloaminopeptidase[END_REF] . It is characterized by hydrophobic residues (Gln317, Val459, Met462, Glu572, Tyr575, Met1034) [START_REF] Mcgowan | Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase[END_REF] that can accommodate the benzylmalonyl substituent of 2 or larger substituents [START_REF] Flipo | Novel Selective Inhibitors of the Zinc Plasmodial Aminopeptidase PfA-M1 as Potential Antimalarial Agents[END_REF] . The introduction of a fluorine atom at this position, although tolerated, is not critical for activity (compound 1 vs 8 and 9, Table 1). Interestingly, a T-staking [START_REF] Bertrand | The Crystal Structures of TrkA and TrkB Suggest Key Regions for Achieving Selective Inhibition[END_REF][START_REF] Whitehead | Human HDAC isoform selectivity achieved via exploitation of the acetate release channel with structurally unique small molecule inhibitors[END_REF] between Tyr575 and phenylmeth-(Z)-ylidene moeity is observed (Figure 1), explaining why the Z isomer ( 2) is more active than the E isomer (3).

The amide function is engaged in two hydrogen bonds with Gly460 and Ala461 backbones. The S'1 pocket is also delimited with hydrophobic residues (Thr492, Val493 and Val 523) [START_REF] Mcgowan | Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase[END_REF] . Although a cation-π interaction is observed between the benzyl group of 2 and Arg489, the introduction of the electron-withdrawing fluorine has a positive impact on activity (2 vs 4, Table 1). This may be the result of an increased desolvation entropy upon binding in this pocket. Also, the presence of the positively charged Arg489 explains why pyridine is less tolerated on that position (compounds 13-14, Table 1). Parasite growth inhibition and cytotoxicity: None of the compounds tested was cytotoxic for MRC5 cells (IC50 values >32 μM, Table 2). Compounds 1,2,6-9,11-12 inhibited parasite growth at IC50-values ranging between 11 to 50 µM (Table 2) which is comparable to other PfAM1-selective or mixed PfAM1/LAP inhibitors 12,[START_REF] Skinner-Adams | Identification of Phosphinate Dipeptide Analog Inhibitors Directed against the Plasmodium falciparum M17 Leucine Aminopeptidase as Lead Antimalarial Compounds[END_REF][START_REF] Cunningham | Chemical Target Validation Studies of Aminopeptidase in Malaria Parasites Using {alpha}-Aminoalkylphosphonate and Phosphonopeptide Inhibitors[END_REF][START_REF] Mcgowan | Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase[END_REF] or bestatine analogues [START_REF] Velmourougane | Synthesis of New (-)-Bestatin-Based Inhibitor Libraries Reveals a Novel Binding Mode in the S1 Pocket of the Essential Malaria M1 Metalloaminopeptidase[END_REF] .

However there is a lack of correlation between enzymatic and parasite activities. Compounds very similar in structure, yet displaying low nanomolar to micromolar potencies on the enzymatic assay, show all poor activity on the parasite (ie. 6 vs 1; 10 vs 8). Before the pharmacokinetic evaluation in mice, 2 was further characterized in vitro. 2 displays a Michael acceptor function that may be sensitive to soft nucleophiles like thiols. An in vitro glutathione alkylation study [START_REF] Kassahun | Identification and characterization of the glutathione and N-acetylcysteine conjugates of (E)-2-propyl-2,4-pentadienoic acid, a toxic metabolite of valproic acid, in rats and humans[END_REF] revealed that compound 2 is isomerized in vitro in its (E)isomer 3 through the GSH-adducts production (Z/E : 54/46, after 24 hours). The proportion of combined GSH-adducts of the two unsaturated isomers reaches 50% at 24 hours. 2 was also tested in vitro for mouse microsomal stability and was found to be very stable in a classic 30minute assay. Interestingly, this stability was observed for both the oxidative and the non-oxidative metabolism, suggesting that no hydrolysis of the hydroxamate moiety occurs through esterase activity.

Pharmacokinetics of 2 in mice: Female CD1 mice received 2 at 50 mg/kg intraperitoneally to evaluate whether it would be concentrated enough for in vivo activity against P. berghei infection. As the parasite is located in erythrocytes, both the plasma concentration and the distribution in erythrocytes were investigated. 2 was formulated in 50/50 DMSO/PBS at 10 mg/mL. Blood was collected at regular time intervals after dosing (Figure 2). Plasma and red blood cells (RBCs) were fractionated and quantification of 2 was performed using LC/MSMS. Concentrations in plasma () or red blood cells () found in female CD1 mice receiving single IP administration at 50 mg/kg. Symbols represent averages from two mice. Plasma and red blood cell data are presented as µmol/L.

Following a single dose, 2 could be measured readily after dosing and until the end of the experiment (six hours post-dose) (Figure 2), showing a plasma elimination half-life of one hour (from 1 to 6 h.; Table 4). As expected from the stability studies, only traces of carboxylic acid were formed, indicating that the hydroxamate was not a primary site of metabolism.

Fortunately, as opposed to in vitro results, no GSH-mediated isomerisation occurred in mice 2 penetrates red blood cells with a significant AUC0→ ∞ of 98 µmol * h/L measured in red cells (Figure 2, Table 4). The global AUC for 2 in red blood cells is even higher than that in plasma suggesting that 2 enters and accumulates in RBCs. Thus the gap between in vitro and in vivo activity could not be explained by a lack of host cell penetration. However, it could possibly be explained by the trapping of the compound by a component of red blood cells before it is able to reach the parasite. It is known that Zn 2+ ion ligands, such as dorzolamide, can be trapped in RBC by carbonic anhydrase [START_REF] Hasegawa | Binding of dorzolamide and its metabolite, Ndeethylated dorzolamide, to human erythrocytes in vitro[END_REF] . Carbonic anhydrase is a zinc metallohydrolase that is located in red blood cells in high concentrations (150 µM total isoforms). Nevertheless, 2 does not inhibit hCAII (0% inhibition at 10 µM and 50 µM, tested in duplicate), indicating that it does not bind carbonic anhydrase and likely is free to enter the parasitic cell from the cytoplasm of red blood cells. In all, this shows that the gap between enzymatic inhibitory potency and antiplasmodial activity cannot be explained by any of the molecular properties of 2 tested by us.

CONCLUSION

Based on literature data, PfAM1 has been regarded as essential to the malaria parasite.

However, none of its described inhibitors, including ours, was able to kill the parasite at concentrations below 1 µM, raising the question of the pharmacological relevance of inhibiting PfAM1 to treat malaria. To formulate a conclusion, we must take into account that the pharmacological activity of an inhibitor is not only determined by its binding to the target and its inhibitory potency, but also by its ability to reach the target at a sufficient concentration for a sufficient period of time. The lack of pharmacokinetic data on inhibitors reported earlier made it difficult to interpret their poor antiplasmodial potency. In this paper, we report the first structurally homogeneous family of inhibitors demonstrating a 100-fold range of enzyme potency, together with antiplasmodial activities and key pharmacokinetic parameters.

Structure-activity relationships established by "fluorine scanning" showed that best position for fluorine is in para of the benzylamido group. Substitution at the malonic carbon is tolerated whereas introduction of a pyridine is deleterious for activity. The range of compounds tested here spanned a 2.5 log scale of inhibitory potency on the isolated enzyme.

However, in this population, we were not able to observe any correlation between enzymatic potency and antimalarial activity. To rule out potential compound-related issues in the course of the validation of PfAM1 as a drug target, we performed stability and distribution studies with 2 (BDM14471), our most promising inhibitor, taking into account potency, physicochemical parameters and metabolic stability. Pharmacokinetic experiments in mice evidenced a significant distribution of 2 in plasma and red blood cells until 6 hours after dosing. In this experiment, 2 reached micromolar concentrations within the red blood cells that harbour the parasites in culture. Altogether, these results show that the compound reaches the site of action but fails to kill the parasite at concentrations relevant to the enzymatic inhibitory potency, suggesting that killing the parasite remains a challenge for potent and drug-like catalytic-site binding PfAM1 inhibitors. A working hypothesis to explain these results could be that the catalytic activity of the PfAM1 is not critical for the survival of the parasite at the intra-erythrocyte stage. In this situation, other non-proteolytic regulatory functions of PfAM1 may have to be targeted to produce antimalarial drug candidates.

MATERIALS AND METHODS

General chemistry. 1 H NMR and [START_REF] Gardiner | Plasmodium falciparum: new molecular targets with potential for antimalarial drug development[END_REF] C NMR were recorded at room temperature on a

Bruker™ DPX 300 at 300 and 75 MHz, respectively. Chemical shifts are in parts per million (ppm). The assignments were made using one dimensional (1D) 1 H and [START_REF] Gardiner | Plasmodium falciparum: new molecular targets with potential for antimalarial drug development[END_REF] C spectra or twodimensional (2D) HSQC and COSY spectra. Mass spectra were recorded on a LCMS system (Waters). HPLC analysis were performed using a C18 TSK-GEL Super ODS 2 µm particle size column, dimensions 50 * 4.6 mm. LCMS gradient starting from 100% H2O / 0.1% formic acid and reaching 20% H2O / 80% MeOH / 0.08% formic acid within 5 min at a flow rate of 1 mL/min was used. Purity (%) was determined by reversed-phase chromatography HPLC using UV detection (215 nm) , and all compounds showed purities greater than 95%. All commercial reagents and solvents were used without further purification.

Coupling reactions for amide bond formation: a) Carboxylic acid (0.66 mmol; 1 eq.) was dissolved in DCM (7 mL) and TEA (102 µL; 0.73 mmol; 1.1 eq.) was added. The mixture was cooled at 0 °C (ice bath) and ethylchloroformate (70 µL; 0.73 mmol; 1.1 eq.) was added dropwise. The reaction mixture was stirred 30 min. at 0 °C, then the desired amine (0.73 mmol; 1.1 eq.) was added. The organic layer was stirred 1h at room temperature and then evaporated. The crude product was dissolved in ethyl acetate, washed twice with aq.

NaHCO3 5%, once with water and once with aq. NaCl, dried over MgSO4, filtered and evaporated. The product was purified by thick layer chromatography (DCM 100%) and/or precipitated in petroleum ether (40-60) to give the expected amide. Or b) Carboxylic acid (0.1M/DMF; 1 eq.), DIEA (2.4 eq), EDCI (1.1 eq.) and HOBt (1.1 eq.) were stirred 5 min at room temperature. Then the appropriate amine (0.1 M/DMF; 1 eq) and DIEA (2 eq.) were added. The mixture was stirred 5 h at room temperature and then solvents were removed under reduced pressure. The residue was dissolved in AcOEt and the organic phase was washed with aqueous NaHCO3 5% (6 times) and NaCl (once). The organic layer was dried over MgSO4 and evaporated under reduced pressure. The residue was precipitated in petroleum ether.

Tert-butyl deprotection. Tert-butylated intermediate was dissolved in a suspension of BTFA (0.75 M/TFA; 25 eq.) with 0.4% H2O. The reaction mixture was stirred 4 h to 5.5 h at room temp. Solvents were removed under reduced pressure and the residue was dissolved in water. The aqueous phase was basified with NaOH 1 M to pH = 7 and extracted three times with AcOEt. The organics layers were dried over MgSO4, filtered and evaporated. The residue was precipitated in ether-pentane. Yields given are those of the deprotection step.

N-(4-Fluoro-benzyl)-N'-hydroxy-2-[1-phenyl-meth-(Z)-ylidene]-malonamide (2).

White powder; Yield 37% (overall); Purity 99%; NMR

N-(4-Fluoro-benzyl)-N'-hydroxy-2-[1-phenyl-meth-(E)-ylidene]-malonamide (3).

White powder; Yield 50% (overall); Purity 99%; NMR

N-Benzyl-2-[1-(4-fluoro-phenyl)-meth-(Z)-ylidene]-N'-hydroxy-malonamide (10).

Beige powder; Yield 15%; Purity 99%; NMR PfAM1 inhibition. Native PfAM1 was purified according to the procedure described by Allary et al. [START_REF] Allary | Properties, stage-dependent expression and localization of Plasmodium falciparum M1 family zinc-aminopeptidase[END_REF] , and diluted 10 times in Tris-HCl buffer (25 mM; pH 7.4) before use. The assays were set up in 96-well plates. The compounds were tested at the concentration of 10 µM. 33 µL of purified PfAM1 were pre-incubated 10 min at room temperature with 33 µL of the inhibitor (30 µM in Tris-HCl buffer, 0.3% DMSO). 33 µL of the substrate Leu-pNA (Km = 0.099 mM) 0.3 mM in Tris-HCl buffer were then added. The reaction kinetics performed at room temperature was followed on a UV-microplate reader MultiskanRC (Labsystems, Finland) at 405 nm. The control activity was determined by incubating the enzyme in the same conditions without inhibitor. Bestatin was used as the reference inhibitor (IC50=284 nM). The statistical Z' factor for the test was 0.82, allowing activities to be determined with a single point with a 95% confidence. Initial velocities are expressed in µmol. min -1 . Data were normalized to the controls that represent Vmax. For the determination of IC50s, initial velocities were plotted as a function of inhibitor concentration, using XLfit software from IDBS. µL of purified APN were pre-incubated 10 min at room temperature with 33 µL of the inhibitor (30 µM in Tris-HCl buffer, 0.3% DMSO). 33 µL of the substrate Leu-pNA (Km = 0.099 mM) 0.3 mM in Tris-HCl buffer were then added. The reaction kinetics performed at room temperature was followed on a UV-microplate reader MultiskanRC (Labsystems, Finland) at 405 nm. The control activity was determined by incubating the enzyme in the same conditions without inhibitor. Bestatin was used as the reference inhibitor (IC50 = 2.7 µM). The statistical Z' factor for the test was 0.75, allowing activities to be determined with a single point with a 95% confidence. [START_REF] Kassahun | Identification and characterization of the glutathione and N-acetylcysteine conjugates of (E)-2-propyl-2,4-pentadienoic acid, a toxic metabolite of valproic acid, in rats and humans[END_REF] Initial velocities are expressed in µmol. min -1 . Data were normalized to the controls that represent Vmax. For the determination of IC50s, initial velocities were plotted as a function of inhibitor concentration, using XLfit software from IDBS.

N-(4-Fluoro-benzyl)-2-[1-(4-fluoro-phenyl)-meth-(Z)-ylidene]-N'-hydroxy

In vitro P. falciparum culture and drug assay. Chloroquine-resistant P. falciparum The following mobile phase gradient was applied: 0-100% (B) in 7.30min; hold at 100% (B) for 1min; 0-100% (A) in 0. This analysis was performed using a software developed at CEREP,SA.

Pharmacokinetic study [START_REF] Peng | Separation and identification methods for metalloproteinase inhibitors[END_REF] BDM14471 was formulated in 50/50 DMSO/PBS at 10 mg/mL and solubilized by short ultrasonic treatment (bath) if necessary. Twelve female CD-1 mice (approx. body weight 25-30 g) were given 1 x 50 mg/kgsingle administration by intraperitonal injection and 2 mice for negative control (no treatment (blank plasma)). Food and drinking water remained available ad libitum throughout the trial. Blood samples (2 mice per data point) were collected at 15 min, 30 min, 1 h, 2 h, 4 h and 6 h. The blood was collected intracardially at the stated time point using a heparinized syringe (1 droplet -2% heparin) and transferred into an Eppendorf tube. The tubes were put on ice and centrifuged at 2600 rpm for 10 minutes to separate the plasma. The fractions of red cells or plasma were stored at 4 °C before analysis. Oasis®-HLB 1cc (30 mg) flangeless cartridges from Waters.Inc were activated with 1 mL methanol, then equilibrated with 1 mL water MilliQ.

Samples were treated as follows. To 100 µL of plasma were added 2 µL of internal standard and 400 µL of water. To 100 µl of red blood cells were added 400 µL of water. The resulting suspension was sonicated and centrifuged; 400 µL of the supernatant were collected and 2 µL of internal standard were added. The resulting mixtures (from plasma or red blood cells) were eluted on an HLB-column. The column was then washed with 0.25 mL of water/MeOH (95/5 v/v). The compounds were eluted with a solution of acetonitrile/water (95/5 v/v) containing 0.1% of formic acid. The eluate was evaporated and re-dissolved in 200 µL of a methanol/mobile phase 60/40 (v/v) for LCMS-MS analysis.

Scheme 2 .

 2 Scheme 2. Synthesis of PfAM1 inhibitors 6-14.(a) i. 1.1 eq. ethylchloroformate, 1.1 eq. TEA, DCM, 30 min., 0 °C; ii. 1.1 eq. R1-CH2-NH2, 1h, room temp.; (b) R1-CH2-NH2, EDCI/HOBt, DMF/DIEA, 5 h, room temp. (c) BTFA (0.75 M/TFA; 25 eq.) with 0.4% H2O, room temp. for t-butyle; (d) TFA 2%/DCM, TIS, 10 min, room temp. for trityle; (e) KOH, EtOH abs., overnight, room temp., (f) i. 1.1 eq. ethylchloroformate, 1.1 eq. TEA, DCM, 30 min., 0 °C; ii. 1.1 eq. H2N-OTrt. HCl, 1 h, room temp.

  (nM) : 284 ; b IC50 bestatine (nM) : 2700 ; c mean of 3 experiments , standard error below 10% ;

Figure 1 .

 1 Figure 1. Binding of 2 in PfAM1.(A) Docking of 2 in the enzyme (PDBcode : 3T8V). Zinc ion is colored magenta. The atoms oxygen, nitrogen are colored red and blue respectively, fluorine is colored green and carbon is shown in light pink or light gray for respectively 2 or key residues of PfAM1. Specific hydrogens are colored white. (B) Schematic representation of the interactions of 2 with PfAM1, featuring the 3-letter code of amino-acids lining the binding pocket. Polar residues are pink (blue circles for positively charged, red circles for negatively charged), apolar residues a re green. Metal contacts are represented as black dotted lines. Hydrogen bonds in blue (acceptor) or green (donor) arrows respectively, Arene-H and arene-positive charge are represented as green dotted lines.

Figure 2 .

 2 Figure 2. Concentration-time profile for compound 2.

  3 was not detected in the in vivo experiment either in plasma or in red blood cells.

Trityl deprotection.

  Tritylated intermediate was dissolved in TFA 2%/DCM and triisopropylsilane was added drop by drop until the yellow color disappeared. The reaction mixture was stirred 5 min. at room temperature, solvents were removed under reduced pressure and the residue was washed with ether-pentane. Yields given are those of the deprotection step.

  pAPN inhibition. Microsomal neutral aminopeptidase (APN) from porcine kidney was purchased from Sigma Inc. as an ammonium sulfate suspension (3.5 M (NH4)2SO4 solution containing 10 mM MgCl2) 10-40 units/mg protein. The enzyme suspension was diluted 600 fold in Tris-HCl buffer (25 mM; pH = 7.4) before use. The assays were performed in 96-well plates. The compounds were tested at the concentration of 10 µM. 33

  30 min; 100% (A) hold for 10 min. The injection volume was 10 µL and the flow rate of 1 mL/min. Approximately 30% of the eluent was introduced into the triple quadrupole mass spectrometer source. The source temperature of the mass spectrometer was maintained at 300 °C, the declustering potential was 50 V, and the curtain gas pressure was 1.5 mTorr. Collision energy and observed transitions were respectively individually optimized for each compound (MRM mode).Docking.The docking of BDM14471 in PfAM1 (using PDB structure 3T8V) was performed using MOE 2009.10 software from Chem Computing Group.Inc. Briefly, the protein was prepared within the MOE package using Gasteiger partial charges, and the Generalized Born/Volume Integral Formalism for protonation at pH 7. The protein atoms were kept as rigid and the ligands as flexible. No restraints were used during docking (conformational search using MM Iteration Limit 1000; all other standard parameters were set on default) but the binding modes were chosen based on both the most favored predicted interaction energy with PfAM1 and a proper chelating of the Zinc ion by the hydroxamic function.Solubility. LogD. Plasma stability. a) Solubility: 40 µL of the 10 mM solution in DMSO of the sample were added to 1.960 mL of MeOH or PBS at pH = 7.4. The samples were gently shaked 24 h at room temperature, then centrifuged for 5 minutes and filtered over 0.45 µm filters. 20µL of each solution were added to 180 µL of MeOH and analyzed by LCMS-MS. The solubility was determined by the ratio of mass signal areas PBS /MeOH. b) Log D 40 µL of the 10 mM solution in DMSO of the sample were added to 1.960 mL of a 1/1 octanol / PBS at pH = 7.4 solution. The mixture was gently shaked 2 h at room temperature, then the two phases were separated. 20 µL of each solution was added to 180 µL of MeOH and analysed by LCMS-MS. Log D was determined as the logarithm of the ratio of concentrations of product in octanol and PBS, determined by mass signals. c) Plasma stability. 40 µL of the 5 mM solution in DMSO of the sample were added to 1.960 mL of rat plasma (Male rat from Charles River Laboratories) to obtain a 100 µM final solution. The mixture was gently stirred 96 h at 37 °C. Aliquots of 200 µL were taken at various times (from 0 to 96 h) and diluted with 200 µL of phosphoric acid (0.14 M). 10 µL of the 2 mM solution in methanol of the internal standard were added. Compounds were extracted three times with 2 mL of AcOEt. The organic layer was evaporated, diluted with 200 µL of methanol and quantified by LCMS-MS, using a calibration curve.Microsome stability. All chemicals were obtained from Sigma-Aldrich (Steinheim, Germany). Solvents were from common sources and of HPLC grade. Stock solutions of all compounds were prepared in methanol at a concentration of 0.1 mM. Pooled male mouse (CD-1) liver microsomes were purchased from BD gentest (Le Pont de Claix, France). All incubations were performed in duplicate in a shaking water bath at 37 °C. The incubation mixture were prepared in polypropylene tubes and contained 1 µM test compound (1% methanol), mouse liver microsomes (0.6 mg of microsomal protein / mL), 5 mM MgCl2, 1 mM NADP, 5 mM glucose-6-phosphate, 0.4 U/mL glucose-6-phosphate dehydrogenase and 50 mM potassium phosphate buffer pH 7.4 in a final volume of 1.5 mL. Sampling points were taken at 5, 10, 15, 20, 25, 30, 45 and 60 min and reactions were terminated by adding ice-cold acetonitrile containing 1 µM internal standard (1 vol). The samples were centrifuged for 10 min at 4000 g, 4 °C to pellet precipitated microsomal protein, and the supernatant was subjected to LC-MS/MS analysis. Control incubations were performed with denaturated microsomes with acetonitrile containing 1 µM internal standard and sampling points were taken at 0 min and 60 min (to evaluate the compound chemical stability in the experimental conditions). Quantitation of each compound was achieved by conversion of the corresponding analyte/internal standard peak area ratios in LCMS-MS (MRM mode) to percentage drug remaining, using the T0 ratio values as 100%. In vitro intrinsic clearance (CLint expressed as µl/min/mg) was calculated according to: Clint = (dose/AUC∞), where dose is the initial amount of drug in the incubation mixture (1 µM) and AUC∞ is the area under the concentration versus time curve extrapolated to infinity. The slope of the linear regression from log percentage remaining versus incubation time relationships (-k) was used in the conversion to in vitro T1/2 values by in vitro T1/2 = -0.693/k. GSH sensitivity. In a PP-tube of 1 mL are introduced 10 µL of a 10 µM-solution of compound in acetonitrile with 145 µL of phosphate buffer (0.067 M at pH = 7.4) and 145 µL of glutathione (5 mM/ H2O).The final compound concentration is 330 µM. After 1 h of roomtemperature incubation, samples are analyzed by LCMS to look for the glutathione conjugate. hCA inhibition 41 human-erythrocyte carbonic anhydrase activity was measured by following the reaction kinetics of hydrolysis of 4-nitrophenyl acetate (450 µM) on a UVmicroplate reader at 400 nm, at CEREP,SA. The control activity was determined by incubating the enzyme in the same conditions without inhibitor. Acetazolamide (IC50 = 29 nM) was used as the reference inhibitor. The results are expressed as a percent inhibition of control specific activity (100 -((measured specific activity/control specific activity) x 100)).

Table 1 .

 1 Inhibition of PfAM1, pAPN by compounds 1

-3, 5-13.

	Compound	Structure	PfAM1	pAPN inhibition
			inhibition	IC50 (nM) b,c
			IC50 (nM) a,c

Table 2 .

 2 Inhibition

							R'	O
	R	X	N H	O	O	N H	OH	N H 2	O OH	N H	O	OH
			1,2,6-9,11-13			15
	Compound		PfAM1 a		Parasite			Cytotoxicity
			inhibition	growth b			IC50 (µM)
			IC50 (nM)	IC50 (µM)
	1						27	17.4 (59 f )			> 64.0
	2						6	11.0 (24 f )			32.0
	6					210	24.7				> 64.0
	7					256	37.9				> 64.0
	8					310	36.5				> 64.0
	9					258	41.6				> 64.0
	11						43	30.4				32.2
	12						56	50.1				> 64.0
	13					1050	> 64.0			> 64.0
	bestatine d				478 e	8-21 g				_ c
	hPheP[CH2]Phe d					79 e	24-62 g			_ c
	15 d						43 e	6.4 g				_ c

of parasite growth by compounds 1, 2, 6-9, 11-13, 15.

a native enzyme; b Mean of at least three experiments, K1 strain, IC50 chloroquine (nM): 154; c not determined; d reference 28 or 29; e recombinant enzyme; f FcB1 strain IC50 chloroquine (nM): 110; g 3D7 strain IC50 chloroquine (nM): 10.

To elucidate this large activity gap and the lack of correlation between the in vitro inhibition of PfAM1 and the parasite growth inhibition, in vitro ADME parameters were explored for a set of analogues to select a compound for in vivo pharmacokinetics.

in vitro ADME parameters for 2. Some key physicochemical parameters were measured for the most potent and selective compounds 1, 2 and 11 (Table

3

). Compounds 1,2 have comparable solubilities and LogD while 11 is slightly more hydrophobic and less soluble. All three compounds are stable in human plasma but compound 2 is by far the most stable compound in rodent plasma which is critical for in vivo pharmacokinetics. These stabilities are coherent with previous data on hydroxamate stabilities

[START_REF] Flipo | Hydroxamates: Relationships between Structure and Plasma Stability[END_REF]

.

Table

3

. Solubility, LogD and plasma stabilities for 1,2,11.

a : Solubility and LogD are measured from a DMSO stock solution; b : Half-life plasma stability at 37 °C using LC-MSMS (MRM detection mode).

Table 4 .

 4 Pharmacokinetic parameters of 2 in female mice.

	Matrix	Dose (mg/kg IP)	tlast (h)	AUC0→last (µmol * h/L)	AUC0→∞ (µmol * h/L)	t1/2 (h)
		1*50	6
	plasma			66	68	1.0
	RBCs			90	98	1.5

-Benzylidene-N-hydroxy-N'-pyridin-4-ylmethyl-malonamide (14).

	Yield 91%; Purity 99%; NMR 1 H (DMSO-d6)  ppm : 3.06-3.10 (m; 2H); 3.41 (t; J = 7.7 Hz;
	1H); 4.47 (d; J = 5.7 Hz; 2H); 7.20-7.31 (m; 5H); 7.62 (d; J = 6.0 Hz; 2H); 8.63 (t; J = 5.7 Hz;
	CONH); 8.76 (d; J = 6.0 Hz; 2H); 10.64 (s; CONHO); tR LCMS 2.08 min; MS (MH) + m/z 300.
	2White powder;
	Yield 90%; Purity 98%; NMR 1 H (DMSO-d6)  ppm : 4.58 (d; J = 5.7 Hz; 2H); 7.10-7.56 (m;
	7H); 7.76 (s; 1H); 8.45-8.78 (m; 2H + NHCO); 11.09 (s; CONHO); tR LCMS 2.14 min; MS
	(MH) + m/z 298.

malonamide (11). Beige powder; Yield 15%; Purity 95%; NMR 1 H (DMSO-d6)  ppm : 4.36 (d; J = 5.7 Hz; 2H); 7.12-7.36 (m; 6H); 7.43 (s; 1H); 7.56-7.60 (m; 2H); 8.27 (t; J = 5.7 Hz; NHCO); 9.14 (s; OH); 11.03 (s; CONHO); tR LCMS 4.36 min; MS (MH) + m/z 333. 2-Benzyl-N-(4-fluoro-benzyl)-N'-hydroxy-2-methyl-malonamide (12). White powder; Yield 60%; Purity 99%; NMR 1 H (CD2Cl2)  ppm : 1.24 (s; 3H); 2.99 (s; 2H); 4.34 (s; 2H); 7.00-7.23 (m; 9H). tR LCMS 4.66 min; MS (MH) + m/z 331. 2-Benzyl-N-hydroxy-N'-pyridin-4-ylméthyl-malonamide (13). White powder;

  After incubation, test plates were frozen at -20 °C. Parasite multiplication was measured by the Malstat method. An amount of 100 μL of Malstat reagent was transferred to a new plate and mixed with 20 μL of the hemolysed parasite suspension for 15 min at room temperature. After addition of 20 μL of NBT/PES solution and 2 h of incubation in the dark, the absorbance was spectrophotometrically read at 655 nm (Biorad 3550 UV microplate reader). Percentage growth inhibition was calculated compared to the negative blanks. IC50 values are calculated from the duplicate determinations with relative difference below 25%.

	solvents used were: (A) 0.01% formic acid in water; (B) 0.01% formic acid in acetonitrile.
	2/K 1-strain was cultured in human erythrocytes O + at 37 °C under a low oxygen atmosphere
	(3% O2, 4% CO2, and 93% N2) in RPMI-1640, supplemented with 10% human serum.
	Infected human red blood cells (200 μL, 1% parasitaemia, 2% haematocrit) were added to
	each well and incubated for 72 h. Cytotoxicity test. MRC-5 SV2 cells, human fetal lung fibroblast, were cultivated in
	MEM, supplemented with L-glutamine (20 mM), 16.5 mM sodium hydrogen carbonate, and
	5% FCS at 37 °C and 5% CO2. For the assay, 10 4 MRC-5 cells/well were seeded onto the test
	plates containing the prediluted compounds and incubated at 37 °C and 5% CO2 for 72 h.
	After 72 h of incubation, parasite growth was assessed fluorimetrically by adding resazurin
	for 24 h at 37 °C. Fluorescence was measured using a GENios Tecan fluorimeter (excitation
	530 nm, emission 590 nm). IC50 values are calculated from duplicate determinations with
	relative difference below 25%.
	LC-MS/MS analysis. The LC-MS/MS system consisted in a Varian 1200L (Varian,
	Les Ulis, France) a Prostar 430 autosampler, a (Varian, Les Ulis, France) triple quadrupole
	mass spectrometry equipped with an electrospray ionisation source, with a Prostar 325
	detector. Analytes in incubation mixtures were separated by HPLC using a gelTSK C18
	Super-ODS, 5 µm, 50 x 4.6 mm column (Interchim, Montluçon France). The mobile phase

resulted in a malono-hydroxamic druggable hit, 1

. Chemical modifications explored the

ACKNOWLEDGMENTS

The authors thank Dr. N. Willand and B. Villemagne for their help with docking and visualization software. We are grateful to Inserm, Université de Lille2 and Institut Pasteur de Lille. This work was specifically funded by «Région Nord-Pas-de-Calais» and EC.

; TIS, triisopropylsilane; Trt, trityl.