Closed-loop brain-machine interfaces may help restore the autonomy of amputees and tetraplegic patients. However, additional efforts are needed towards their realworld use with prostheses. Here we have interfaced a highly versatile closed-loop mouse BMI with an online model of a realworld prosthetic arm. We describe this setup and illustrate how it allows to explore the efficiency of different input and output coding strategies given a realistic modelling of the interactions between a commercial bidirectional prosthesis and its environment.

I. INTRODUCTION

Invasive Brain-Machine Interfaces (BMIs) are devices that access in real-time neuronal activity inside the brain in order to execute an action. Beyond research applications, this direct interfacing with the brain constitutes an opportunity to help patients suffering from severe motor damage, including paralysis and amputation cases. The architecture of invasive BMIs combines a neuronal recording device, usually multiple microelectrodes implanted in the brain, with an online analysis of the signal to extract commands that can be passed to an effector. This simple, one-directional strategy has already shown impressive results during the past years, successfully giving control of complex robotic prostheses to patients with heavy disabilities [START_REF] Hochberg | Reach and grasp by people with tetraplegia using a neurally controlled robotic arm[END_REF], [START_REF] Ajiboye | Restoration of reaching and grasping movements through brain-controlled muscle stimulation in a person with tetraplegia: a proof-of-concept demonstration[END_REF].

However, emulating the fast, accurate control seen in natural movements remains a challenge. This is in part because explicit feedback mechanisms are often lacking. Indeed, on top of the eye contact with the prosthesis, the user also needs feedback about non-visual properties, including internal forces or occluded touch [START_REF] Sainburg | Control of limb dynamics in normal subjects and patients without proprioception[END_REF]. This is especially true for manipulation of complex, multi-joint prostheses. Moreover, somatosensory and proprioceptive feedback are of utmost importance to induce embodiment and sense of agency, which in turn impacts motor performance [START_REF] Van Den Bos | Sense of body and sense of action both contribute to self-recognition[END_REF]. Recently, efforts have been made to incorporate somatosensory or proprioceptive-like feedback to BMIs, using approaches ranging from fully biomimetic stimulations of sensory cortices [START_REF] Bensmaia | Biological and bionic hands: natural neural coding and artificial perception[END_REF] to learning-based methods using arbitrary patterns of stimulation [START_REF] Dadarlat | A learning-based approach to artificial sensory feedback leads to optimal integration[END_REF].

We have previously described a closed-loop BMI setup [START_REF] Abbasi | A fast intracortical brain-machine interface with patterned optogenetic feedback[END_REF] based on the spiking activity of neurons recorded by chronically implanted electrodes in the primary motor cortex.

This system includes a sensory feedback using a high-speed projector to generate arbitrary photostimulation patterns over the somatosensory cortex of transgenic mice expressing channelrhodopsin (EMX-Cre;Ai27 strain, [START_REF] Madisen | A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing[END_REF]). But so far this promising bidirectional BMI setup has not been tested in the context of interactions with a real-world prosthesis, although this could potentially challenge the usability of the system.

In particular, one issue that may prevent successful control of current brain-machine interfaces is the inherent delay between the readout of the motor command and the feedback. Motor BMIs, as complex systems, introduce new sources of latencies: an algorithmic delay, which duration depends on the complexity of the transformation between the recorded signal and the command output; and a second delay during the actual control of the prosthesis when it involves an additional driver layer, such as a proportional-integralderivative controller [START_REF] Willett | Improving brain-machine interface performance by decoding intended future movements[END_REF]. Finally, the online production of feedback by the BMI and its transduction into neuronal activity also takes time. These many sources of delays are likely to lead to a steep decrease in performance and learning, particularly in terms of error correction and accuracy [START_REF] Shimada | Decrease in prefrontal hemoglobin oxygenation during reaching tasks with delayed visual feedback: a near-infrared spectroscopy study[END_REF].

Beyond latencies, the control of a real-world prosthesis raises many other challenges, in particular related to the specific geometry of the prosthesis actuators, which constraints the structure of the motor commands as well as of the feedback. Here we describe how we have attached a simulation of a commercial prosthesis to our BMI setup, and how this setup allows us to explore and test solutions to these challenges.

II. METHODS

A. Surgical preparation

All animal experiments were carried out using EMX-Cre;Ai27 mice that express channelrhodopsin 2 in their pyramidal neurons. In these mice, blue light illumination triggers spiking discharges in cortical neurons. We implanted the bidirectional BMI interface in two steps. First, to allow photostimulation of the cortical surface, we implanted a 5 mm diameter optical window over the primary somatosensory cortex (S1). During the same surgery, we also implanted a headpost to hold the mouse in place during the awake experiments. After two weeks of recovery, we used intrinsic imaging to confirm that the representation of whiskers in S1 (the so-called barrel cortex) was indeed located below the glass window. Finally, we implanted the mice with a 32-channel Neuronexus extracellular electrode in the whisker primary motor cortex (M1). After one week of recovery, the mouse was ready to be trained in the BMI task.

Control of a robotic prosthesis simulation by a closed-loop intracortical brain-machine interface

Dorian Goueytes, Aamir Abbasi, Henri Lassagne, Daniel E. Shulz, Luc Estebanez and Valérie Ego-Stengel B. Overview of the system Our BMI system (Fig. 1a) is run by a main "master" software providing a graphical user interface and a hub that connects and streams data between three players: (1) the Neural Signal Processor (Cerebus NSP, BlackRock Microsystem), (2) the V-REP simulation software [START_REF] Rohmer | V-REP: A versatile and scalable robot simulation framework[END_REF], and (3) the DLP projector (Vialux High-speed V7001). The NSP contains a hard real-time computer and performs online spike extraction and sorting from the 30 kHz sampled continuous signal coming from the chronic electrode. This spike sorted data is streamed to the master hub. The DLP projector sends patterned light to a 2.5mm² area of the cortex as highdefinition frames (1024 × 768 pixels) with a 100 Hz refresh rate. Finally, the robotic software models an existing prosthesis, the Jaco2 arm from KINOVA, and simulates the interaction of this robotic arm with a virtual environment. The movements of the robotic arm are driven by the neuronal activity streamed from the NSP, and feedback from the robot controls the patterns of photostimulation sent back to the mouse.

C. Master Software

In order to interface the different components of our system and ensure a proper flow of information during runtime, we developed a application in C++ using the Qt environment. This choice was driven by the need to be compatible with the APIs of all other components of the system, as well as by the aim to maximize performance.

The main thread converts the firing rate into a motor command through a transfer function, monitors the state of the prosthesis and sends the appropriate pattern of photostimulation to the DLP projector. A second thread supports the graphical interface that provides a feedback on the status of the experiment as well as ways to adjust its parameters on the fly.

D. Conversion of spiking rates into torques

The prosthesis includes joints that should be actuated in both directions. Here we aimed to control one joint and ensure that the mouse can move it as easily in one direction as in the other. This type of symmetrical control is hard to derive from spiking activity. To address this challenge we implemented an antagonist control strategy, meaning that the joint was placed under the control of two groups of neurons that "pushed" in opposite direction. This strategy is inspired by a recent publication [START_REF] Neely | Volitional Modulation of Primary Visual Cortex Activity Requires the Basal Ganglia[END_REF] showing that it ensures a nonbiased bidirectional control. We used a "greedy" selection algorithm to automatically select two groups of neurons of similar size and with similar firing rate. Those groups were fixed for the whole behavioural sequence across days. Their activity was dynamically weighted at the beginning of every control session depending on a 10 minutes baseline recording in order to avoid the possible build-up of a bias towards one direction due to day-to-day variability.

The actual command applied on the joint was generated from the spiking of these two groups by convoluting their activity with a 100 ms kernel, then translating these two instantaneous firing rate series into torque values, using a logistic function. The difference between these two opposite torques was finally applied to the elbow joint (Fig. 2). We chose to use a biomimetic torque-based control with the hope that learning would be facilitated in these more physiological conditions. Also, it removed the need to include a driver layer such as a PID, which would be necessary for a speed-based control. Fig. 2. The activity of two groups of neurons is merged, transformed into torques that are applied in opposite direction on the same joint.

E. Interfacing the V-REP robotic modelling software

We used the V-REP native remote C++ API to interface our master software with V-REP. It sends the torque commands to the robotic arm model, and reports to the master software the contacts of the arm with the virtual environment. The communication between the two programs works in a similar manner to a client-remote server system timed with a clock. Every time a torque command was sent from the master software, V-REP executed a 5 ms simulation step and returned the state of the prosthesis (posture, contact with the environment), which was then used by the master software to update the photostimulation feedback.

F. Providing structured feedback to the mouse

The DLP device displays blue light, arbitrary 2D photostimulation patterns at the surface of the cortex, which are updated every 10 ms with a power of 25 mW.mm -2 . This configuration allows to explore a very broad range of feedbacks, depending on the type of prosthesis and on the decision to use or not a biomimetic approach.

Guided by the circular nature of the geometry of the prosthesis actuator, we chose to provide feedback to the mouse in the form of the angular position of the prosthesis joint. We encoded the state of the prosthesis as a rotating bar centered on the barrel cortex, and more precisely on the representation of the C2 whisker as determined by the optical intrinsic imaging. The bar thus followed the position of the joint (Fig. 1b).

III. RESULTS

Here we describe work to expand a mouse closed-loop BMI that we developed previously [START_REF] Abbasi | A fast intracortical brain-machine interface with patterned optogenetic feedback[END_REF]. This brain-machine interface was composed of an invasive readout of the spiking activity in M1, and an artificial sensory feedback via the spatially and temporally structured photoactivation of Channelrhodopsin-expressing neurons in S1. We connected this interface to a robotic simulation engine, V-REP, which simulated a 7-degree of freedom arm, the Jaco2 from KINOVA robotics (Fig. 1). To validate the full setup, we focused on a single axis of the robot, corresponding to the first joint after its attachment base. We implemented the translation of spiking activity into torque signals that were relevant to control the robotic joint. On the sensory side, the angular position of the joint was fed back to the mouse as a structured pattern of cortical activation.

A. Calibration of the closed-loop latency

To evaluate the performance of our closed-loop system, we measured two complementary metrics. First we looked at the end-to-end hardware latency, which is the time between the occurrence of a spike recorded by the electrode and the resulting update of the photostimulation feedback. This measure represents the delay induced by all computations and transmissions within our BMI system, and is independent of the behaviour of the animal or the state of the cortex. Second we measured the pace at which the main thread of the master software operated, by measuring with a photodiode the timing of the transition between consecutive photostimulations.

The end to end latency of the main loop averaged 43.82ms (SD 4.80ms, Fig. 3a), while the loop ticked at an average pace of 12.48ms (SD = 2.07ms, Fig. 3b). These figures are larger than the values obtained in the absence of the simulation (average end-to-end latency 12.3ms, SD 1.03, [START_REF] Abbasi | A fast intracortical brain-machine interface with patterned optogenetic feedback[END_REF]) but they are still an improvement compared to the values obtained in some motor only protocols, as discussed in the introduction of [START_REF] Willett | Improving brain-machine interface performance by decoding intended future movements[END_REF].

B. Control of the robotic arm

Two mice were connected to our closed-loop BMI during 5 days, for 30 minutes two times per day (mean 57.5 trials/session, mean trial duration 31.33s). The mice were first habituated to the head fixation, with the BMI apparatus disconnected, for 1 session. Then, during BMI sessions, the activity of units in M1 controlled the rotation of the joint of the Jaco2 robot closest to the arm base. Fig. 4a displays how the spiking activity of five isolated neurons controlled one joint of the simulated Jaco² prosthesis while receiving the real-time feedback. In this particular experiment, one group of 2 neurons generated the positive torque while another group of 3 neurons generated the negative torque. The firing rate of each group was smoothed by a 100 ms box kernel before being fed to the transfer function and generating torques. The torques were then subtracted and used as the final output torque applied to the joint (mean output torque 8.483 kg.m².s -2 across all sessions). Importantly, when we looked at the movement of the arm generated by the animal, we found that the activity generated by the neurons recorded in M1 was sufficient to entrain rotations of the joint, which covered its full 360° span, in the two mice that were involved in the experiments (Fig. 4b).

Overall, those observations illustrate the feasibility and functionality of interfacing our fast BMI with a robotic modelling simulation, and they show the possibility to train surgically implanted mice to drive the prosthetic arm through the BMI.

IV. DISCUSSION

We recently developed a fast bidirectional mouse BMI that incorporates a rich photoactivation-based feedback [START_REF] Abbasi | A fast intracortical brain-machine interface with patterned optogenetic feedback[END_REF].

Here we report on a system that makes this BMI interact with an online simulation of a robotic arm. The mice controlled one joint of the arm using their primary motor cortex activity, and the BMI provided a dynamic feedback about the angular position of the arm by delivering optogenetic stimulation to the primary somatosensory cortex. The full system operated properly and at low latency that will hopefully greatly facilitate learning and performance in our future experiments.

To develop this design, we chose to use V-REP for simulating the robotic actuator because it provides both adaptability and a physically accurate environment. Disadvantages compared to the in-house development of a C++ routine that would simulate the arm dynamics include programming constraints and a potential slowdown of the loop. However, V-REP offers a mature and versatile modelling environment that would be hard to fully replicate. The environment it emulates has driven technical decisions that will remain relevant when connecting the BMI loop to a physical prosthesis. This includes the choice of a joint control based on antagonist groups of neurons, and the choice of a rotating bar feedback. In addition, the networking constraints in V-REP are close to those encountered when working with real-world prosthesis, and addressing them early in our work will help us to seamlessly move from a virtual to a real actuator in the future. This setup will be instrumental to systematically explore the extremely large space of input and output parameters that characterize invasive closed-loop BMIs. On the feedback side, our setup should allow to test different types of feedback structure beyond the rotating bar presented here, as well as different cortical locations. For example, we hope to determine whether biomimetic vs non biomimetic feedback may be optimal for information delivery about the current state of a robotic limb. Also, the suitability of primary sensory cortices vs. higher-order and associative cortical areas could be tested. The same type of approach could be applied to the neuronal spiking readout, by comparing a strategy based on decoding of the spiking activity with a strategy based on adaptive learning of the neuronal networks. In particular, we are interested in determining the impact, on motor control and learning, of additional latencies that would be artificially injected in the BMI loop. Moreover despite the fact that we explored so far only a one degree of freedom task, both our control strategy and feedback structure were designed to scale-up to a higher number of degrees of freedom. This will allow us in the future to explore complex, high-dimensional motion within our BMI framework.

Fig. 1 .

 1 Fig. 1. (a) Information flow through the different components of the closed-loop BMI and prosthesis model. (b) Example of a feedback photostimulation on the mouse barrel cortex. An illumination bar rotates (arrow) around a fixed point (dark blue cross).Fig.2. The activity of two groups of neurons is merged, transformed into torques that are applied in opposite direction on the same joint.

Fig. 4 .

 4 Fig. 4. (a) Example of the transformation of the raw spiking of two antagonist groups of neurons into movements of a robot joint. (b) Distribution of the position of the joint controlled by the BMI during the last five training sessions in two habituated mice. Starting angle of the joint is either 90°, 0° or 270°.Fig. 3. (a) End-to-end latency of the BMI closed-loop. (b) Pace of the loop.

ACKNOWLEDGMENT

The authors would like to thank Patrick Parra for his help with the mechanical work, Guillaume Hucher and Aurélie Daret for her help with the animals.

* Research supported by ANR Neurowhisk; by Labélisation FRM DEQ20170336761 and by the iCODE project funded by the IDEX Paris-Saclay, ANR-11-IDEX-0003-02. All authors are with Unité de Neurosciences, Information et Complexité, Centre National de la Recherche Scientifique, FRE 3693, 91198 Gif-sur-Yvette, France


Sophie Klaimy

Carmen Ciotonea

Jérémy Dhainaut

Sébastien Royer

Mathilde Casetta

Sophie Duquesne

Grégory Tricot

Jean-François Lamonier

Flash Catalytic Pyrolysis of Polyethylene over (Alumino)silicate Materials

Keywords: aluminosilica, zeolite, flash catalytic pyrolysis, polyethylene 10.1002/cctc.201901819

come

Introduction

There has been an over-increasing demand for plastics during the past 50 years due to their versatile uses, low cost and fabrication capabilities, resulting in an always increasing amount of plastic waste generated [1]. Plastic waste has very low biodegradability and requires multiple costly steps namely sorting, washing, grinding and extrusion, for its effective recycling, arising worrisome environmental, social and economic issues. Therefore, it is urgent to find and implement effective and cheaper solutions for the reduction, reconditioning or recycling of the plastic waste [2]. Among the various processes to valorize the plastic waste, the thermal pyrolysis under neutral gas results in a wide distribution of products among them being highly valuable molecules such as aromatics or alkanes [3]. In the absence of oxygen, the thermal decomposition of polymers and of polyolefins more precisely occurs following a complex mechanism involving the formation of free radicals from the random scission of polymer chains [4]. The radical formation can be observed by ESR analysis of pyrolysed samples [4c,d]. The thermal pyrolysis results in the production of three different families of products can be used as fuels and/or sources of chemicals: C1 to C3 gases, liquid hydrocarbons and solid residue [5]. The proportion and composition of the three phases are dependent on reaction parameters such as the temperature and pressure applied, and the type of plastic waste used in the pyrolysis process [6]. While high amounts of plastic waste can be treated following this process, the required high temperature and costs associated to steps of separation refrain the large scale development of the process [7]. To address these drawbacks, catalysts are frequently used in combination with the pyrolysis process, resulting in the decreasing of the temperature necessary for the decomposition of the polymers. Moreover, catalysts are known for their ability to activate and orientate the chemical transformation of polymers [8]. This ability is strongly linked to the catalyst properties, which can be finely tailored depending on the application [9]. In catalytic pyrolysis (or pyrocatalysis), the morphology, the composition as well as the structural and textural properties of the catalysts, all have a notable influence on the conversion mechanism [10]. Therefore, even for simplified plastic waste feeds, such as pure polyethylene, the control and orientation toward selected molecules is challenging and requires a good knowledge of all the parameters involved, regarding the reaction process, but also from the point of view of the catalyst properties. In the literature, a wide variety of heterogeneous catalysts have been reported for catalytic pyrolysis and among them are conventional solid acids such as zeolites, silica-alumina and alumina, but also mesostructured catalysts such as Al-MCM-41 [10,11]. Indeed, the acidity of the catalyst is an important parameter to consider in pyrolysis. In particular, it has been shown that, in the pyrolysis reaction of polystyrene, the surface acidity of the catalyst can be related to the increasing number of initiation sites for the pyrolysis reaction [5]. In this regard, acid catalysts allow to produce higher liquid yields (C5-C9), owing to the breaking of C-C bonds with preferential chain length [11]. An increase in branched alkanes and aromatics has also been observed, while the decomposition temperature and activation energies of decomposition are decreased by comparison with the thermal pyrolysis process [12]. Different mechanisms have been proposed, in which the decomposition of polymers (including polyethylene) follows an ionic mechanism, with the abstraction of the hydride ion occurring over Lewis acid sites,

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and the protonation of olefins formed by thermal decomposition over Brønsted acid sites [13]. Therefore, to achieve a high selectivity in liquid products over the final products of catalytic pyrolysis, the strength (strong and weak), the nature (Lewis or Brønsted and their ratio), and the location of the acid sites have to be finely controlled during the formulation of the catalyst [12]. The ZSM-5 zeolites (MFI-type) are by far the most used in the pyrolysis process. This is related to the combination of favorable features, in particular a good thermal stability and an adjustable acidity (through the silicium over aluminum ratio). Commercial ZSM-5 zeolites are frequently used for studies, but the reported performances do not afford the establishment of clear correlation between catalyst characteristics and product selectivities obtained by pyrocatalysis. This is related to the use of ZSM-5 coming from different suppliers or batches having, in most of the cases, even for comparable acidity, different physical properties [10a,15]. Another important parameter is indeed the catalyst porosity, controlling accessibility of the reactants to the internal active sites and diffusion of products. In the case of zeolites, more than 90% of the acidic sites are located within the crystals and are therefore only accessible through sub-nanometer porosity [16]. The catalytic pyrolysis is then deemed to occur mostly at the surface of the crystals. Other effective catalysts are silicaalumina [17]. Silica-alumina has the advantage of presenting not only acid sites but also larger pore sizes than zeolites. Consequently, during the decomposition process, in a first step larger molecules are cracked to smaller molecules by a primary reaction, leading to the diffusion of these molecules within the pores of the catalyst where further reactions can proceed leading to the production of non-aromatic products. Considering the lower acidity developed by amorphous silica-alumina, limited cracking occurs, and isomerization is promoted [18]. This work describes the polyethylene (PE) conversion over (alumino)silica catalysts under Flash Catalytic Pyrolysis conditions with the objective to discriminate the role of catalyst main properties (acidity, specific area, particle size and pore size distribution) on the product distribution and liquid product characteristics. Compared to classical (catalytic) pyrolysis process, flash (catalytic) pyrolysis is characterized by a high heating rate and short residence time of products at high temperature, conditions affording to maintain high liquid yields [19]. Materials synthesized include silicic and aluminosilicic MFI-type zeolites (Silicalite-1 and ZSM-5), in addition to amorphous SiO2 and silica-alumina. The results demonstrate the interest of the process for the recycling of plastic waste, with the possibility, varying the catalyst properties, to orientate reaction from paraffin-olefin to cycled hydrocarbon-aromatic.

Results and Discussion

Characterization of catalysts

X-ray diffractograms of the synthetized materials are shown in Figure 1 (a). Si-MFI and Si/Al-MFI show the typical reflections of MFI structure by comparison with the JCPDS pdf file # 44-003. The absence of a broad peak at 2 = 20-25 ° confirms the production of highly crystalline materials even after the ionic exchange for the Al-rich zeolite for counter-ion exchange. For Si-MFI, the presence of low quantity of quartz phase is evidenced by the presence of a peak at 2θ of 22° (Figure S1). Quartz formation is associated with the harsh conditions applied for the synthesis (high temperature and long period of hydrothermal treatment) [20]. The XRD pattern of Si/Al and SiO2 (not shown) exhibits only a broad signal in the 2θ range 20-25° which is characteristic of amorphous silica [11]. Figure 1( b) presents the N2 physisorption isotherms of materials. The isotherms registered over the MFI zeolites are characterized by a well-defined plateau, according to Type I shape of the IUPAC classification. A sharp adsorption step occurs at low relative pressures (P/P0 < 0.1) which is indicative of the formation of a microporous material. For both amorphous materials (SiO2 and Si/Al), the registered isotherms are of Type IV, which is classically encountered for porous materials with broad pore size distribution in the mesopore domain [14]. Table 1. Textural properties for SiO2, Si/Al, Si-MFI and Si/Al-MFI materials.

SBET [a] m 2 g -1 S [b] m 2 g -1 Sext [c] m 2 g -1 Vp [d] cm 3 g -1

V [e] cm 3 g -1 Dpart [f] Textural properties are displayed in Table 1. BET specific surface areas of 387 m 2 .g -1 and 402 m 2 .g -1 for Si/Al-MFI and Si-MFI, respectively are obtained, with a major contribution from the micropores (Sµ being >85% of the total surface area).

10.1002/cctc.201901819

Accepted Manuscript

ChemCatChem

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Besides, the value of the total pore volume is close to that of the microporous volume, confirming that most of the porosity of the solids is related to the micropore structure. In contrast, for both amorphous materials, the contribution of the microporous surface to the BET surface is negligible. Surface areas of 140 and 222 m 2 .g -1 are obtained for Si/Al and SiO2, respectively. When comparing silica or alumina-silica containing samples, the total pore volume developed by the amorphous materials is 4 times larger than that of the crystalline solids. On the contrary, the micropore volumes are 12 to 17 times larger for MFI-type materials than for amorphous solids (Table 1).

Figure 2 shows SEM images illustrating the differences of morphology between the two MFI samples. Si-MFI crystals present a coffin shape (Figure 2 DLS analysis was performed in order to determine the particle size distribution (Figure 3). The two MFI samples are showing large average diameter, with a maximum at 1800 nm for Si/Al-MFI and 2400 nm for Si-MFI. These sizes are directly comparable with the average sizes of the objects observed in SEM images (Figure 2 The presence of extra-framework aluminum species in hexahedral coordination is evidenced by the peak at 0 ppm [22a]. Signal integration finally allows quantifying these two Al sites, with 90% of [4] Al and 10% [6] Al species. Presence of Al-OH linkages was then monitored with the editing of 2D 1 H/ 27 Al correlation maps. As previously reported, the correlation signals observed here are signature of Al-OH linkages owing to the very short recoupling time used in the pulse sequence [22b]. The 2D map recorded on the Si/Al sample indicates that the three Al species are involved in Al-OH site but comparison between the 1D and the 2D Map 27Al projection (Figure 4, and horizontal axis of Figure 5a) suggests that [4] Al sites are less involved in the Al/H interaction than the two higher coordination states. A single correlation signal can be observed in the 2D map acquired on the Si/Al-MFI sample showing the presence of [4] Al-O-H linkages. No signal involving the extra-framework [6] Al can be found in the 2D map but the low intensity of this signal has to be taken into account. Quantity (mmol.g -1 of desorbed NH3) and relative strength of the acid sites (temperature of NH3 desorption) were obtained by temperature programmed desorption of ammonia (NH3-TPD). The NH3-TPD profiles are shown in Figure 6 and the corresponding quantitative information are provided in Table 2. Regardless of their structural and textural characteristics, the two pure silica materials, SiO2 and Si-MFI, have a low acidity, with a total quantity of NH3 desorbed below 0.05 mmol.g -1 .

These two materials are distinguished by the presence of a weak acidity for SiO2 which can be related to silanol presence on its external surface [22a]. As expected, the addition of aluminum to silica generates acidity. This extra acidity is related to (i) Brønsted acidity which resides in the bridging hydroxyl group originating from the replacement of Si 4+ in the framework by Al 3+ and (ii) Lewis acidity assigned to the presence of extraframework alumina species [22b]. Si/Al presents a total NH3 desorption of 0.209 mmol.g -1 mostly assigned to weak acidity with two desorption peaks located at 210 °C and 290 °C. Si/Al-MFI exhibits a much higher density of acid sites, characterized by a high total NH3 desorption of 0.638 mmol.g -1 . Two NH3 desorption peaks are clearly distinguished (Figure 7). The first peak is centered at around 250 °C and corresponds to weak acidity (0.193 mmol.g -1 ), as observed for the Si/Al catalyst. The second peak is centered at around 420 °C and corresponds to strong acid sites (0.445 mmol .g -1 ). Such sites are not observed over Si/Al catalyst for which the second desorption occurs at a temperature 150 °C below.

Pyrolysis of polyethylene

Thermogravimetric study of the decomposition

Figure 7 shows the TG curves obtained during PE thermal and catalytic pyrolysis. In the case of PE thermal decomposition, the weight loss is observed to start at 420 °C, with a complete decomposition observed at 502 °C. The use of pure silica catalysts (SiO2 and Si-MFI) lead to a decomposition of PE at a temperature identical to the thermal decomposition showing the complete inefficiency of these two solids as catalysts to promote the decomposition of PE at lower temperature. The residual weight however differed depending on the solid used for the Samples Temperature [a] (°C)

Acidity [b] (mmol.g -1 )

LT peak

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experiment. The higher residual weight obtained for SiO2 suggests the formation of higher wax proportion retained on the solid than using Si-MFI. When Si/Al-MFI is used, a slight shift of the decomposition temperature toward the lower temperature is observed, with a decomposition starting at 400 °C and ending at 490 °C. Then, the Si/Al-MFI catalyst is able to activate decomposition of PE at lower temperature than the thermal decomposition temperature. In this case, the maximum mass loss is close to 90 %, demonstrating the formation of a very low proportion of residual wax than when pure silicas are used. Finally, with Si/Al catalyst, the temperature shift is far more marked than using Si/Al-MFI. The starting decomposition of PE is measured at 300 °C, with the decomposition ending at 460 °C.

The residual weight, even if less important than using pure silica sample, remains higher than over Si/Al-MFI. Then Si/Al is more prone to form waxy compounds which are not decomposed at high temperatures than Si/Al-MFI. Based on the temperature at which 50% of PE is degraded, the following rank can be established: Si/Al > Si/Al-MFI > Si-MFI ~ SiO2 ~ thermal. The comparison of Si/Al and Si/Al-MFI results suggests also that strong acid sites, mainly located in the micropores of the zeolite, are not needed for activating the PE decomposition. Si/Al exhibits a much higher external surface area than Si/Al-MFI (140 m 2 .g -1 vs 7 m 2 .g -1 ). Therefore a high external surface area with sufficient concentration of acid sites seems to be adapted to activate the PE decomposition at low temperature [12].

Catalytic flash pyrolysis

Catalyst effect on the phases distribution: Figure 8 presents the distribution in gas, liquid, wax and residue at the end of the flash pyrolysis reaction (40 min at 450 °C). The low activity of pure silica materials (Si-MFI and SiO2) is characterized by an incomplete decomposition of PE (4 wt.% of residue with SiO2; 23 wt.% of residue with Si-MFI), and with a consequent formation of wax (31 wt.% for SiO2 and 29 wt.% for Si-MFI). The slightly better performances obtained with SiO2 can be explained by its higher total acidity (45 mol.g -1 ) in comparison with that of the Si-MFI (17 mol.g -1 ), in addition to its four times higher external surface area. Even when using a catalyst without acidity, a radical-activated mechanism is expected to occur, the pore structure promotion (i.e. a larger surface available for polymer-solid contact) allows to explain the slightly better results obtained with SiO2 [24]. In line with the thermogravimetric study, Si/Al and Si/Al-MFI are the most active catalysts for the PE pyrolysis since in both cases, the PE decomposition is complete and no residue remains at the end of the experiment. Moreover, waxy compounds are not observed when using Si/Al-MFI, while less than 10% are measured with Si/Al. those results are consistent with previously discussed TG results. The gas and liquid yields increases then significantly, with liquid yields reaching 35-36 wt.% with Si/Al and Si/Al-MFI. The significant improvement in liquid+gas yield obtained using the Si-Al containing catalyst is a clear evidence of the need of acidic sites to activate the decomposition of PE, according to a carbenium ion-activated mechanism instead of radical-type mechanism [16]. Similarly to our results, Lin et al. reported that the decomposition of PE using ZSM-5 led to higher gas yield than when silica-alumina catalyst is used [15]. The increase in gas phase proportion observed for Si/Al-MFI can then be related to the presence of strong acid sites, sites that can initiate cracking reactions over heavy compounds. [25]. This evolution is also in line with the higher formation of gas fraction since the highest gas fraction is obtained with Si/Al-MFI. Nature of liquid phase products: Figure 10 further details the proportion of paraffins, olefins, cycled hydrocarbons and aromatics obtained after flash pyrolysis reaction. Flash thermal pyrolysis leads to high yields in paraffins (33 wt.%) and olefins (48 wt.%). These results are in accordance with other authors [26]. Interestingly, in the conditions of flash catalytic pyrolysis, the presence of both SiO2 and Si-MFI allows to promote the cyclization of decomposition products, the crystalline Si-MFI framework leads to a higher proportion of cycled hydrocarbons (34 wt.%) than with SiO2 (25 wt.%) while only 6 wt.% of cyclic molecules are obtained under thermal conditions. Both aluminosilicate catalysts lower the fraction of paraffinic + olefinic compounds when compared to the pure silica counterparts. A significant fraction of aromatics (25 wt.%) in addition to cycled hydrocarbons (51 wt.%) is obtained in the presence of Si/Al-MFI. On the contrary, Si/Al promotes formation of cycled hydrocarbons (38 wt.%) and olefins fraction remains consequent (45 wt.%). As previously mentioned, the primary products issued from the PE cracking forms on the external surface acid sites of the Si/Al and Si/Al-MFI materials. Lin et al also observed the formation of a higher proportion of olefinic C3-C7 compounds with Si/Al than with ZSM-5 [17], which is perfectly in line with the presented results. The promotion of aromatization (as BTX [18]) and cyclization observed in the case of Si/Al-MFI (or ZSM-5), when compared to Si/Al can be explained by transformation of primary light products on strong acid sites located in the microporosity. Therefore, while the use of a catalyst of mild acidity is preferred to obtain linear molecule, catalysts with strong acidic sites afford the production of valuable aromatics.

Conclusions

Different (alumino)silicate catalysts with controlled properties (size, morphology, acidity) were prepared, characterized, and used for the flash catalytic pyrolysis of polyethylene (PE). Based on thermogravimetric analysis, catalytic decomposition of PE is activated in the presence of silica-alumina (amorphous or MFItype) catalyst. Using pure silica containing material (amorphous or MFI-type) did not allows to convert PE at lower temperature than using thermal conditions. These results were confirmed during the flash catalytic pyrolysis of PE experiments, performed at 450 °C. Amorphous Si/Al and MFI-type Si/Al containing zeolite are the most efficient catalysts, and they allow the complete decomposition of PE with a limited formation of wax. On the contrary, poorly acidic silicic catalysts present a low decomposition activity, resulting in the formation of a large amount of residue and wax. The liquid to gas phase distribution is significantly affected by the catalyst acidity, a higher gas phase proportion being obtained when the catalyst acidity increases. In addition, the acidity increase leads to an increase of the light molecule fraction in the liquid phase (C3 to C5). From a nature molecule point of view, the strong acidity exhibited by the Si/Al MFI-type zeolite favors the formation of aromatics and cycled hydrocarbons, while the lowest acidity displayed by the amorphous aluminosilicate favors olefins formation.

Experimental Section

Chemicals: All chemicals were used as purchased: sodium hydroxide (NaOH 98 wt.%, Alfa Aesar), sodium aluminate (NaAlO2 98 wt.%, Alfa Aesar), Ludox HS40 (SiO2 40 wt.%, Aldrich), ammonium nitrate (NH4NO3 98 wt.%, Alfa Aesar), Tetraethylorthosilicate (TEOS 98 wt.%, Acros), Tetrapromylammonium hydroxide (TPAOH 1 M in water, Sigma), Aluminium isopropoxide (Al(OiPr)3 98 wt.%, Sigma Aldrich), ethanol 10.1002/cctc.201901819

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(EtOH 99.9%, Alfa Aesar), linear low density polyethylene (Eltex® PF6220AE) from Wipak (Bousbecque, France). SiO2 sample was kindly provided from Saint Gobain (SS61138).

Silicalite-1 preparation:

A solution composed of H2O, TEOS and TPAOH is prepared with the following sol composition 100 SiO2: 400 EtOH : 40 TPAOH : 2000 H2O. The mixture was prepared at room temperature and aged for 24 h under stirring. The pH was adjusted to 11 by adding 10 wt.% sodium hydroxide solution. The resulting solution was transferred in a Teflon-lined autoclave and crystallized at 140 °C for 48 h under static conditions. The template removal was performed by calcination for 2 h at 200 °C (2 °C min -1 ), then for 8 h at 550 °C (0.5 °C min -1 ) under air [11a]. An ion exchange process was carried out in the presence of 0.5 M NH4NO3, for 12 h at 70 °C. The ion exchange step is followed by a calcination at 500 °C for 3 h (0.5 °C min -1 ) under air to produce the H-form zeolites. The final sample was denoted Si-MFI.

ZSM-5 preparation. Preparation involves two successive steps. The first step is for seeds synthesis. Seed suspension was prepared from a mixture of sodium hydroxide, H2O and TPAOH. After complete dissolution, Ludox is added dropwise and the mixture is stirred for 1 h at room temperature. Mixture molar composition is 100 SiO2 : 20 Na2O : 6.6 TPAOH : 4750 H2O. Hydrothermal treatment was performed at 100 °C for 16 h, and the resulted seeds were kept in solution form.

Thereafter, in a second step, a solution composed of H2O, sodium hydroxide and sodium aluminate is prepared and kept at room temperature under stirring for 1 h. Then the silica precursor is added and the solution is maintained under stirring at room temperature for 1 h, with the following molar composition of 1.87 Al2O3 :100 SiO2 : 4 Na2O : 2500 H2O. Finally, the seed suspension is added dropwise to the reaction mixture without any purification. Hydrothermal treatment was performed at 180 °C for 40 h under static conditions. The removal of the organic template was carried by calcination (2 h at 200 °C (2 °C min -1 ), followed by 8 h at 550 °C (0.5 °C min -1 ) under air [START_REF] Lechert | Verified synthesis of zeolitic materials[END_REF]). After the ion exchange process (see preparation of Si-MFI sample), the sample was denoted Si/Al-MFI.

Amorphous aluminosilica sample. The sol composition for the preparation of the amorphous aluminosilica sample was 100 SiO2: 1150 EtOH : 1000 H2O : 6.2 Al2O3, according to the procedure described in [11a]. The sol was aged at 60 °C for 24 h, then calcined at 550 °C for 3 h (5 °C min -1 ) under air. The sample was denoted Si/Al.

Sample characterization: Sample characterization involves ICP, XRD, N2-physisorption, SEM, DLS particle size measurement, 27 Al and proton solid state NMR, NH3-TPD, and ATG study of PE decomposition. X-ray diffraction was performed on a Bruker AXS D8 Advance diffractometer in Bragg-Brentano configuration fitted with a LynxEye Super Speed detector. XRD patterns were recorded with CuKα radiation (λ = 0.154 nm, 40 kV, 30 mA) in the 3-60° 2θ range with a 0.02° 2θ step. N2physisorption isotherms were recorded at -196 °C on a Tristar II Plus system from Micromeritics Instruments. Before analysis, the samples were outgassed under dynamic vacuum at 300 °C for 5 h. The textural properties were determined from the adsorption/desorption isotherms by using the MicroActiveTristar II Plus software version 5.02. The BET surface area was determined using the multipoint BET algorithm in the P/P0 range of 0.10-0.25 (criteria: correlation coefficient > 0.998 ; C constant > 0 ; Rouquerol transform increases on the selected portion). The t-plot method was applied to quantify the micropore volume and surface area (de Boer statistical thickness of 3.8-6.5 Å). Scanning Electron Microscopy analysis was performed on a JEOL JSM-7800F LV instrument equipped with a FEG source. Prior to the analysis, the samples were metalized with a layer of 180 nm of chromium. The particle size was measured by using ImageJ software. The average hydrodynamic diameters of calcined materials were determined by Dynamic Light Scattering (DLS, Nano ZS from Malvern Instruments). DLS was applied with an angle of 173 by using He-Ne laser (4 mW) operated at 633 nm. For the analysis, the materials were dispersed in water under ultrasound (3 min). 27 Al MAS-NMR experiments were performed at 208.5 MHz on an AVANCE III 18.8T spectrometer equipped with a 3.2 mm probe head operating at spinning frequencies of 20-22 kHz. The quantitative spectra were acquired with a 1 s pulse length, 512 transients and a recycle delay of 0.5 s. The [x] Al quantification was directly obtained by signal integration. The 2D 27 Al/ 1 H map was obtained with the Dipolar-Heteronuclear Multiple-Quantum Correlation NMR technique (D-HMQC) [START_REF] Tricot | [END_REF]. The 4096 x 120 acquisition points were recorded with a SR4 2 1 recoupling scheme of 200 s. Each slice was acquired with 192 transients and a recycle delay of 1s. Temperatureprogrammed NH3 desorption (NH3-TPD) experiments were carried out on a Micromeritics Autochem II 2920 chemisorption analyser instrument. Ammonia concentration in the outlet mixture was monitored using an OmnistarTM Pfeiffer mass spectrometer. Prior to the desorption, the samples were outgassed under helium at 200 °C (60min, 10 °C min -1 ). Sample saturation is performed under 10 % NH3 in He (30 mL min -1 ) at 100 °C (130°C for Si/Al-MFI sample) for 30 min. The desorption experiment was performed with a ramp of 10 °C min -1 from 100 °C to 700 °C. ATG measurements of PE decomposition were carried out on a SDT Q600 TA instrument. The experiments were performed under a N2 flow of 100 mL min -1 applying a temperature ramp of 10 °C min -1 up to 900 °C. The catalyst loading is fixed at 10 wt.%.

Flash (Catalytic) Pyrolysis tests: thermal and catalytic pyrolysis reactions of PE were carried out in a pyrolysis pilot composed of a homemade reactor, a furnace (CARBOLITE CERGO 30-3000 °C) and a liquid condensation system. The reactor is described in Figure 12. It is composed of a stainless steel tube (length of 700 mm, diameter of 60.3 mm). Catalyst weight (10 wt.%) was mixed with the PE and placed in the cooling coil coupled to a chiller (LAUDA ECO SILVER) where the temperature was controlled at 20±1 °C. The reactor was then purged under nitrogen (5 L.min -1 ) for 5 min, and the flow was stabilized at 200 mL.min -1 . Flash pyrolysis was carried out by introducing the sample in the heated zone after temperature stabilization at the reaction temperature. As the pyrolysis can lead to the formation of a liquid phase, a gas phase and a solid residue, the liquid phase composed of condensable molecules was recovered in two baths of liquid nitrogen and the residue remains in the sample holder. Wax product can also be produced, and recovered at the exit of the reactor. At the end of the experiment, liquid, solid and wax were quantified. The gas molecule yield was calculated by subtracting the liquid/wax and residue amount from the initial polymer weight. Liquid phase analysis was performed by GC-MS using a shimadzu GC-2010 coupled to a Shimadzu GC-MS-QP2010 plus mass spectrometer.

The GC column is a 30m*0.25µm*0.25 µm optima-5ms capillary column.

In addition, the oil was separately analysed for quantification using GC-201 Shimadzu equipped with an FID detector and a 30*0.32 mm*0.25 µm Zebron ZB-5 capillary column for separation. The protocols applied are detailed in Table 3.

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Figure 1 .

 1 Figure 1. (a) XRD patterns for Si-MFI and Si/Al-MFI materials (bottom: ZSM-5 (MFI) reference, JCPDS pdf file # 44-0003); (b): N2 physisorption obtained for SiO2, Si/Al, Si-MFI and Si/Al-MFI.

  (a-b)), which is typical for crystalline MFI materials[21]. The average object sizes measured from SEM images are around 3.8 µm in length and 0.8 µm in thickness showing the formation of a microcrystalline material. Interestingly, Si/Al-MFI is presenting a completely different morphology, described as raspberry-shaped particulates (Figures 2d, FigureS1(a-b))[10a, 13]. The objects formed are of homogenous diameter, around 1 µm, and are composed of aggregated nanocrystals with an average size of 80-90 nm, as illustrated in the FigureS1(b). The nanocrystals are expected to form during the first step of the synthesis (corresponding to the seeds synthesis), while the second step of the synthesis results in their aggregation to the raspberrytype objects.

Figure 2 .

 2 Figure 2. SEM images recorded for SiO2, Si/Al, Si-MFI and Si/Al-MFI catalysts.

Figure 3 .

 3 Figure 3. Particle size distribution obtained by DLS measurement for SiO2, Si/Al, Si-MFI and Si/Al-MFI materials.

  (b, d)). A sharper distribution is obtained for SiO2 and Si/Al materials, with a maximum measured in the 60-70 nm interval. The particle size distribution obtained by DLS analysis is again in good agreement with the particle size observed on SEM images (FiguresS3).

Figure 4 .

 4 Figure 4. 1D 27 Al MAS-NMR spectra acquired at 18.8 T on the Si/Al and Si/Al-MFI samples.

Figure 5 .

 5 Figure 5. 27 Al( 1 H) D-HMQC spectra recorded at 18.8 T with a very short recoupling time of 200 s on the Si/Al (a) and Si/Al-MFI (b) samples. The 2D maps are accompanied by the 27 Al and 1 H projections in the horizontal and vertical axis.

Figure 6 .

 6 Figure 6. NH3-TPD profiles obtained for SiO2, Si/Al, Si-MFI and Si/Al-MFI. Si/Al-MFI TCD signal is divided by 100 for the sake of comparison.

  temperature for NH3 desorption. [b] quantity of NH3 desorbed 10.1002/cctc.201901819 Accepted Manuscript ChemCatChem This article is protected by copyright. All rights reserved.

Figure 7 .

 7 Figure 7. Thermogravimetric analysis for SiO2, Si/Al, Si-MFI and Si/Al-MFI materials and pure PE.

Figure 8 .

 8 Figure 8. Phase distribution obtained during flash pyrolysis of PE reaction at 450 °C. Blue = liquid, red = wax, yellow= gas, and green = residue.

Figure 9 .

 9 Figure 9. Products distribution of the liquid from the decomposition of PE at 450 °C. Blue = C3 to C5, black = C6 to C9, burgundy = C10+.

Figure 10 .

 10 Figure 10. Composition of liquid product from the decomposition of PE at 450 °C. Blue = paraffins, orange = olefins, grey = cycled hydrocarbons, purple = aromatics, and green = others.

Figure 12 .

 12 Figure 12. Scheme of the pyrolysis pilot.

Table 2 .

 2 NH3-TPD analysis for the aluminosilicate materials.

Table 3 .

 3 GC protocol applied for liquid phase analysis.

	Carrier gas	He	N2
	Carrier gas flow	24.2 mL min-1	14.3 mL min-1
	Initial temperature/ initial time	40 °C	40 °C
	Heating rate	8 °C min-1	8 °C min-1
	Final temperature/ final time	280 °C	280 °C
	Injection temperature	300 °C	300 °C
	Split flow	20 mL min-1	10 mL min-1
	Transfer line	300 °C	-

GC-MS

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-Page No. Flash catalytic pyrolysis of polyethylene over (alumino)silicate materials

  Table of Contents (Please choose one layout) series of amorphous and crystalline (alumino)silicate catalysts, with variable acidity, are evaluated for the flash pyrolysis of polyethylene (PE).

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	Sophie Klaimy, Carmen Ciotonea,
	Jérémy Dhainaut, Sébastien Royer,
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	Grégory Tricot and Jean-François
	Lamonier*
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Acknowledgements

This work is part of the PYROCAT project co-financed by the European Union with the European Regional Development Fund (ERDF). Chevreul Institute (FR 2638), Ministère de l'Enseignement Supérieur et de la Recherche and Région Hauts-de-France are also acknowledged for supporting this work. The authors acknowledge Sophie Heymans from Neo-Eco Development for her participation to the work. The authors thank Olivier Gardoll, Laurence Burylo and Alexandre Fadel for their contribution in NH3-TPD, XRD and SEM analysis, respectively.


A, B D Eugénie Romero

Saoussen Oueslati

Mohamed Benchekroun

Agathe C A D'hollander

Sandrine Ventre

Kamsana Vijayakumar

Corinne Minard

Cynthia Exilie

Linda Tlili

Pascal Retailleau

Agustin Zavala

Eddy Elisée

Edithe Selwa

Laetitia A Nguyen

Alain Pruvost

Thierry Naas
email: thierry.naas@aphp.fr.

‡,‖,Ʇ, ¶,◊ Bogdan I Iorga
email: bogdan.iorga@cnrs.fr.

Robert H Dodd

Kevin Cariou
email: kevin.cariou@cnrs.fr

Azetidinimines as a novel series of non-covalent broad-spectrum inhibitors of β-lactamases with submicromolar activities against carbapenemases of classes

The rise of resistances in Gram negative bacteria is reaching an extremely worrying situation and one of the main causes of resistance is the massive spread of very efficient β-lactamases, which render most β-lactam antibiotics useless. Herein, we report the development of a series of imino-analogs of β-lactams (namely azetidinimines) as efficient non-covalent inhibitors of β-lactamases. Despite the structural and mechanistic differences between serine-βlactamases KPC-2 and OXA-48 and metallo-betalactamase NDM-1, all three enzymes can be inhibited at a submicromolar level by compound 7dfm, which can also repotentiate imipenem against a resistant strain of Escherichia coli expressing NDM-1. We show that this compound

can efficiently inhibit not only the three main clinically-relevant carbapenemases of Ambler classes A, B and D, but also β-lactamases of all four classes (A, B, C and D). Our results pave the way for the development of a new structurally original family of non-covalent broadspectrum inhibitors of β-lactamases.

TABLE OF CONTENTS

The discovery of penicillin was the start of a golden age for antibiotherapy that is now threatened by exponentially increasing antibioresistance phenomena. [START_REF] O'neill | Tackling drug-resistant infections globally: final report and recommendations[END_REF] Because β-lactams (Figure 1a) have been, and still are, the most prescribed antibiotics worldwide, resistance against them is particularly alarming. Gram negative bacteria (GNB), the main mechanism of β-lactam resistance is due to the production of ß-lactamases, enzymes capable of hydrolyzing β-lactams. Even carbapenems, the most powerful β-lactams, are not spared by metallo-β-lactamases (MBLs) such as NDM-1, IMP-1 or VIM-1 (class B) and/or by some clinically worrisome serine β-lactamases (SBLs) such as KPC-2 or OXA-48 (classes A and D, respectively). [START_REF] Nordmann | Superbugs in the coming new decade; multidrug resistance and prospects for treatment of Staphylococcus aureus, Enterococcus spp. and Pseudomonas aeruginosa in 2010[END_REF][START_REF] Nordmann | Global spread of carbapenemase-producing enterobacteriaceae[END_REF] The discovery of novel antibiotics acting on novel targets is difficult to foresee and strategies to overcome β-lactam resistance [START_REF] Worthington | Overcoming resistance to β-lactam antibiotics[END_REF][START_REF] Chellat | Targeting Antibiotic Resistance[END_REF] especially via enzyme inhibition [START_REF] González-Bello | β-Lactamase Inhibitors To Restore the Efficacy of Antibiotics against Superbugs[END_REF][START_REF] Tooke | β-Lactamases and β-Lactamase Inhibitors in the 21st Century[END_REF] may preserve our current therapeutic arsenal. This strategy was implemented more than 30 years ago with the development of β-lactamase inhibitors (BLIs) including clavulanic acid or sulbactam (Figure 1b), but was not actively pursued until recently. Since 2012 several new broad-spectrum inhibitors of class A and class C β-lactamases have emerged. Of significant interest, avibactam [START_REF] Ehmann | Avibactam is a covalent, reversible, non-β-lactam β-lactamase inhibitor[END_REF][START_REF] Wang | The road to avibactam: the first clinically useful non-β-lactam working somewhat like a β-lactam[END_REF] and vaborbactam [START_REF] Hecker | Discovery of a cyclic boronic acid β-lactamase inhibitor (RPX7009) with utility vs class A Serine carbapenemases[END_REF] (Figure 1b) were approved by the FDA for clinical use whilst many of their congeners, in particular diazabicyclooctanes, [START_REF] Hirsch | In vitro activity of MK-7655, a novel β-lactamase inhibitor, in combination with imipenem against carbapenem-resistant Gram-egativne bacteria[END_REF][START_REF] Morinaka | OP0595, a new diazabicyclooctane: mode of action as a serine β-lactamase inhibitor, antibiotic and β-lactam 'enhancer[END_REF][START_REF] Livermore | In vitro activity of cefepime/zidebactam (WCK 5222) against Gram-negative bacteria[END_REF][START_REF] Durand-Réville | ETX2514 is a broadspectrum β-lactamase inhibitor for the treatment of drug-resistant Gram-negative bacteria including Acinetobacter baumannii[END_REF][START_REF] Peilleron | Synthetic approaches towards avibactam and other diazabicyclooctane β-lactamase inhibitors[END_REF] are currently going through preclinical or clinical development. These compounds are mainly able to efficiently inhibit SBLs, including carbapenemases, of class A, C and sometimes D, but generally do not inhibit MBLs (class B). In parallel, continuous efforts for the development of efficient MBL inhibitors [START_REF] Linciano | Ten years with New Delhi metallo-β-lactamase-1 (NDM-1): from structural insights to inhibitor design[END_REF] have recently led to the identification of promising molecules that can bind to zinc atoms of the active site of MBLs, such as thiols [START_REF] Tehrani | Thiol-containing metallo-β-lactamase inhibitors resensitize resistant Gram-negative bacteria to meropenem[END_REF] -including the clinically available antihypertensive agent L-captopril [START_REF] Klingler | Approved drugs containing thiols as inhibitors of metallo-β-lactamases: strategy to combat multidrug-resistant bacteria[END_REF] -, aspergillomarasmine A, [START_REF] King | Aspergillomarasmine A overcomes metallo-β-lactamase antibiotic resistance[END_REF] rhodanines [START_REF] Xiang | Rhodanine as a potent scaffold for the development of broad-spectrum metallo-β-lactamase inhibitors[END_REF] and their thienolate derivatives [START_REF] Brem | Rhodanine hydrolysis leads to potent thioenolate mediated metallo-β-lactamase inhibition[END_REF] or heteroaryl-carboxylic acids such as ANT431 [START_REF] Leiris | SAR studies leading to the identification of a novel series of metallo-β-lactamase inhibitors for the treatment of carbapenem-resistant enterobacteriaceae infections that display efficacy in an animal infection model[END_REF] (Figure 1c). Yet there is still no MBL inhibitor available for clinical use. Moreover, the emergence of bacterial isolates producing two or even three different carbapenemases of different classes now dictates the development of inhibitors capable of simultaneously inhibiting SBLs and MBLs. So far, apart from some polyphenolic derivatives with moderate activities, [START_REF] Yu | Virtual target screening reveals rosmarinic acid and salvianolic acid A inhibiting metallo-and serine-βlactamases[END_REF] only boronic acids have been reported to efficiently inhibit both SBLs and MBLs (Figure 1d). Rigid cyclic analogues of vaborbactam such as VNRX-5133 (currently in phase 3 clinical trials) were the first molecules shown to inhibit all classes of β-lactamases (Figure 1d). [START_REF] Brem | Structural basis of metallo-β-lactamase, serine-β-lactamase and penicillin-binding protein inhibition by cyclic boronates[END_REF][START_REF] Cahill | Cyclic boronates inhibit all classes of β-lactamases[END_REF][START_REF] Krajnc | Bicyclic boronate VNRX-5133 inhibits metallo-and serine-β-lactamases[END_REF][START_REF] Parkova | Broad spectrum β-lactamase inhibition by a thioether substituted bicyclic boronate[END_REF] VNRX-5133 exhibits submicromolar inhibitory activity against most MBLs and SBLs, being only slightly less active against IMP-1 (class B) and OXA-48 (class D). Very recently, thiol/boronic acid hybrids (such as MS18) were developed and demonstrated interesting dual inhibition properties against SBLs and MBLs. [START_REF] Wang | Structure-based development of (1-(3′-mercaptopropanamido)methyl)boronic acid derived broad-spectrum, dual-action inhibitors of metallo-and serine-β-lactamases[END_REF] MS18 and its congeners also possess a rather broad scope but show limited effects against NDM-1 (class B) and OXA-48.

In this context, we sought to develop novel inhibitors that could block the activities of both SBLs and MBLs with comparable efficiency, specifically targeting the three most clinicallyrelevant carbapenemases: KPC-2 (class A), NDM-1 (class B) and OXA-48 (class D).

We addressed this challenge by exploring uncharted chemical space around the β-lactam nucleus. Synthetically, this four-membered ring can be obtained by a [2+2] cycloaddition between a ketene and an imine, [START_REF] Pitts | Chemical synthesis of β-lactams: asymmetric catalysis and other recent advances[END_REF] a reaction named the Staudinger synthesis after its discoverer (Scheme 1a). [START_REF] Staudinger | Zur Kenntniss der Ketene[END_REF] We recently reported that carefully substituted ynamides [START_REF] Dekorver | Ynamides: A modern functional group for the new millennium[END_REF][START_REF] Evano | Ynamides: Versatile tools in organic synthesis[END_REF] can be used as precursors for the in situ generation of ketenimines [START_REF] Lu | The thriving chemistry of ketenimines[END_REF][START_REF] Dodd | Ketenimines Generated from Ynamides: Versatile Building Blocks for Nitrogen-Containing Scaffolds[END_REF] under mild conditions, which can be intercepted by various heterocyclic nucleophiles [START_REF] Hentz | Transition-metal-free tunable chemoselective N-functionalization of indoles with ynamides[END_REF] or can undergo a microwave assisted [2+2] cycloaddition with imines. [START_REF] Romero | Base-Mediated Generation of Ketenimines from Ynamides: Direct Access to Azetidinimines by an Imino-Staudinger Synthesis[END_REF] By replacing the ketene (C=C=O) with the in situ generated iminoketene (C=C=NR), the reaction directly led to an azetidinimine (Scheme 1b).

Such imino-β-lactams have only been rarely studied with respect to their synthesis [START_REF] Van Camp | Synthesis of N-(tosyl)azetidin-2-imines[END_REF][START_REF] Whiting | Copper-catalyzed reaction cascade: direct conversion of alkynes into N-sulfonylazetidin-2-imines[END_REF] and never with respect to their biological and particularly, antibiotic activities. Taking into account their structural similarities with β-lactams, we thus decided to evaluate their therapeutic potential against carbapenemases.

RESULTS AND DISCUSSION

Chemistry. Taking into consideration the scope and the fact that only aryl groups can be easily incorporated in our previously developed methodology, [START_REF] Romero | Base-Mediated Generation of Ketenimines from Ynamides: Direct Access to Azetidinimines by an Imino-Staudinger Synthesis[END_REF] we devised a convergent synthetic plan to access as many structural variations as possible on the azetidinimine scaffold. The Ar 1 group originated from aniline 1x, which was protected by a Boc group to give 2x (Scheme 2).

Coupling of 2x with brominated triisopropylsilylacetylene provided ynamide 3x after TBAFpromoted desilylation. Condensation of benzaldehyde 4y with aniline 5z afforded imines 6yz. Scheme 2. General synthetic plan to prepare tri-arylated azetidinimines 7xyz.

The key [2+2] cycloaddition was performed under microwave heating and gave azetidinimines 7xyz with concomitant loss of the Boc protecting group. For para-methoxy derivatives 7aaa and 7aba, as well as para-benzyloxy derivative 7dfc, their corresponding para-phenols 7aam, 7abm and 7dfm were obtained by ether cleavage using BBr3 (for OMe) or AlCl3 (for OBn). More than forty compounds were prepared using this route.

First results. The first compound evaluated was 7aaa, bearing a p-anisyl group on the endocyclic nitrogen. This aryl group was initially chosen for synthetic reasons as it would electronically favor the reaction and could also be easily derivatized. Initial assessment showed that in the presence of 10 µM of 7aaa, the hydrolysis of imipenem by NDM-1 was inhibited by 90% with an IC50 estimated to be between 2 and 5 µM (Table 1). This very encouraging initial result prompted us to try and rationalize this inhibitory activity by performing in silico molecular modeling studies (Figure 2). It appeared that the azetidinimine does not behave like a β-lactam, whose carbonyl group is chelated by the zinc ions during the hydrolysis process. Here, the methoxy group is coordinated by both zinc ions in the active site and the four-membered ring acts as a scaffold to position the two phenyl rings in hydrophobic regions of the enzyme active site. The Ar 1 phenyl substituent is surrounded by Ser251, Asp212, Ala 215 and Ser217 for both enantiomers, whereas the Ar 2 phenyl substituent is positioned near Asn220 in the R enantiomer and in the proximity of Val73, Ile 35, Met67 and Phe70 in the S enantiomer (Figure 2). The two enantiomers of 7aaa were separated by chiral chromatography and then tested separately, both showing similar inhibition of NDM-1.

SAR for enzyme inhibitory activities.

A systematic structure-activity relationship was then undertaken by varying the aromatic groups around the azetidinimine nucleus and targeting three carbapenemases: NDM-1, OXA-48 and KPC-2. In addition to its NDM-1 inhibitory activity, 7aaa was found to be moderately active against KPC-2 but did not have any activity against OXA-48 (Table 1). Variations of the Ar 3 ring showed that minimal modifications such as replacing the methoxy of 7aaa by an ethoxy (7aab) or a benzyloxy (7aac) led to an improvement of the activity against all three enzymes, 7aac in particular displaying a submicromolar activity against NDM-1 (0.4 µM). Similar activities to 7aab and 7aac were observed when the methoxy group was in the meta position (7aad). However, the presence of two methoxy groups (7aaf), or a benzo[d] [1,3]dioxole moiety (7aag), led to a slight decrease of efficiency against NDM-1 and a loss of activity for OXA-48 and KPC-2. This was partially restored by incorporation of a 2,3-dihydrobenzo[b] [1,4]dioxane, as 7aah was found to be quite similar to 7aad in terms of inhibition profile. While 3,4,5-trimethoxy derivative 7aai only maintained a low activity for NDM-1, aniline-derived compound 7aaj was found to inhibit all three enzymes with IC50's of 10.0, 7.0 and 3.5 µM, respectively. The incorporation of an iodine atom (7aak) on the para position led to solubility issues preventing data from being obtained against OXA-48 and KPC-2 although the anti NDM-1 activity could nevertheless be evaluated to be in the 4.0-5.0 µM range. The thiomethyl derivative 7aal was also poorly soluble in water, but its strong affinity for the active site Zn ions led to a high inhibition of NDM-1. Finally, the free phenol 7aam was moderately active against KPC-2 and OXA-48 but again exhibited high inhibitory properties against NDM-1 (0.8 µM). To conclude in this series of Ar 3 variations, the best pan-carbapenemase inhibitors were p-oxyphenyl compounds and especially those bearing a benzyloxy (7aac) and a free hydroxy (7aam). Keeping a para-methoxyphenyl group as Ar 3 , variations of Ar 2 were then examined for activity and compared to 7aaa (Table 2). The p-chloro analogue (7aba) maintained a similar activity against NDM-1 and though some anti-OXA-48 activity was witnessed, its anti-KPC-2 effect was largely lost. Both p-methoxy and p-fluoro analogues 7aca and 7ada were rather inefficient on all three enzymes while the p-iodo, 3,5-dichloro and 2-naphthyl derivatives (7aea, 7aga and 7aha respectively) presented an interesting profile, being almost equipotent against NDM-1 and KPC-2 but somewhat less effective against OXA-48. In contrast, pyridinyl compound 7aia

was almost inactive on all three enzymes. From these results, two Ar 2 groups were selected: para-chloro and 2-naphthyl (as in 7aba and 7aha). At this stage, we wished to evaluate the influence of Ar 1 in both series (4-chloro in Table 3 and 2-naphthyl in Table 4). It is worth noting that 4-chloro derivatives generally exhibit a greater ease of synthesis due to the superior reactivity of imine 6bz (or 6fz) in the [2+2] cycloaddition. Compared to 7aba, the introduction of a substituent at the para position of Ar 1

-whether a chloro (7bba), a bromo (7cba), an iodo (7dba), a trifluoromethyl (7eba) and to a lesser extent a methoxy group (7fba) -was highly beneficial for the anti-NDM-1 activity, with all compounds possessing IC50 values in the 0.5-0.8 µM range (Table 3). The IC50's against KPC-2 were also improved with values between 2.0 and 3.6 µM, except for (7fba). Additionally, the IC50's against OXA-48 dropped under the 10 µM threshold for two compounds (7cba and 7dba). A similar trend could be observed with an additional chloro at the 2 position of Ar 2 (2,4-dichloro series): the para-iodo (7dfa) and para-trifluoromethyl (7efa)

were found to be the most potent pan-inhibitors of carbapenemases NDM-1, OXA-48 and KPC-2 in this series. Similarly, excellent anti-NDM-1 activities were observed in the Ar 2 = naphthyl series (Table 4), with four compounds (7dha, 7bhb, 7dhb and 7fhb) having submicromolar IC50's. Activities against KPC-2 and OXA-48 were also significantly improved, being always under 10 µM except in the case of para-chlorinated derivatives 7bha and 7bhb (for OXA-48). Despite their high activity, the increased lipophilicity brought by the naphthyl group (for 7dhb logPtheor = 6.94) caused products in this series to be very poorly soluble and to easily form aggregates in the assay media. Thus further studies within this series were stopped.

Having witnessed the highly favorable effect of the para substitution on Ar 1 and keeping in mind the importance of the para-alkoxy moiety on Ar 3 , further study of the influence of Ar 2

was carried out with 7dba as reference (Table 5). All compounds in this series offered a rather broad-spectrum of activity with most of them inhibiting all carbapenemases with an IC50 below 10 µM and even submicromolar for NDM-1. In contrast with the other compounds, p-fluoro (7dda), p-iodo (7dea) and 3,5-dichloro (7dga) analogues were more active against OXA-48 than against KPC-2. Finally, keeping Ar 1 = p-iodophenyl and Ar 2 = 2,4-dichlorophenyl (7dfa), variations on Ar 3 were then further investigated. Surprisingly (compared with 7aaa and 7aac

in Table 1), the p-benzyloxyphenyl derivative (7dfc) proved to be less active than 7dfa (Table 5). 7dfc was then converted into a free phenol (7dfm) by AlCl3-mediated cleavage of the ether bond (see Scheme 3). This compound exhibited submicromolar IC50 activity against the three enzymes: 0.1 µM for NDM-1 (which could be explained by stronger interactions of the OH function with the zinc ions of the active site), 0.4 µM for OXA-48 and 0.6 µM for KPC-2. While the homologous benzylic alcohol analogue 7dfn was found to be less active, the corresponding carboxylic acid 7dfo (obtained by oxidation of 7dfn with KMnO4, see Scheme 3) was the most active compound on NDM-1 so far (0.07 µM) but with a complete loss of activity against OXA-48. The metabolic stability of 7dfm was evaluated and the compound was found to possess an excellent stability profile with a Clint of µL/min/mg protein in mouse hepatic cells (Table 7).

The toxicity of 7dfm was measured against both normal cells (MRC-5) and cancer cells (HCT-116) and was found to be in the 20-30 µM range, that is, almost two orders of magnitude higher than its IC50 against carbapenemases. Finally, compound 7dfm was evaluated for the repotentiation of imipenem against the clinical strain of E. coli that expresses NDM-1 (amongst other genes of resistance). [START_REF] Bonnin | Characterization of an IncFII Plasmid Encoding NDM-1 from Escherichia coli ST131[END_REF] In the absence of inhibitor the MIC was above the resistance threshold at 16 µg/mL, in the presence of 20 µM of 7dfm it was divided by 2, while using 50 µM of 7dfm it was divided by 4, bringing it down to the intermediate/resistant limit. Molecular modeling Compound 7dfm was selected for a more in depth study of the interaction with the three main clinically-relevant carbapenemases, NDM-1, KPC-2 and OXA-48. The docking of these three enzymes with the two enantiomers of 7dfm was performed using GOLD [START_REF] Verdonk | Improved protein-ligand docking using GOLD[END_REF] and the results are presented in Figure 4. With NDM-1, both enantiomers of 7dfm interact in a similar manner as 7aaa (see Figure 2).

The phenol moiety is coordinated with the two zinc ions and the 4-iodo-phenyl substituent is positioned in the same subpocket in both cases, possibly establishing a stabilizing halogen bond with the side chain of Asp212. The 2,4-dichloro-phenyl substituent is positioned in a hydrophobic environment bordered by residues Val73, Ile35, Met67 and Phe70 for the S enantiomer (Figure 4d) and in the vicinity of Asn220 for the R enantiomer (Figure 4a).

The docking pose of KPC-2 with the R enantiomer (Figure 4b) shows two hydrogen bonds between the phenol group of 7dfm and the side chains of Ser70 and Lys73. The 4-iodo-phenyl substituent is positioned in a subpocket formed by the side chains of Asn218, His219 and Glu276 and may form a halogen bond with the side chain of Asn218, whereas the 2,4-dichlorophenyl substituent is more solvent exposed. The S enantiomer makes hydrogen bonds with the side chains of Ser70 and Ser130 through the phenol substituent (Figure 4e), and the positions of the other two substituents are inverted compared with the R enantiomer.

The interaction of OXA-48 with the R enantiomer of 7dfm (Figure 4c) shows a hydrogen bond between the phenol group and the side chain of Thr104, at the upper extremity of the binding site, whereas the two other substituents are positioned more deeply in the binding site groove. The S enantiomer establishes the same hydrogen bond between the phenol group and the side chain of Thr104, with the positions of the 4-iodo-phenyl and 2,4-dichloro-phenyl substituents inverted.

Conclusions

The evaluation of azetidinimines, imino-analogues of β-lactams, as β-lactamase inhibitors led to the development of a new family of non-covalent carbapenemase inhibitors. Structural optimization led to the identification of phenol 7dfm as the lead compound, which can strongly inhibit MBL NDM-1 (0.1 µM), class A SBL KPC-2 (0.4 µM), and class D SBL OXA-48 (0.6 µM). Molecular modeling showed that this compound does not mimic a β-lactam within the enzyme active sites and that both enantiomers can interact with the enzymes. Provided one key interaction (such as complexation with the zinc ions in NDM-1 or hydrogen bonds in KPC-2 and OXA-48) is established, the rigid and compact structure of the central four-membered ring indeed allows the other aryl groups to be allocated almost interchangeably in the enzyme pocket to increase the affinity. Further refinement of the structure of these molecules will be pursued to improve the efficiency of these β-lactamase pan inhibitors. The metabolic stability of the hit compound and its efficient repotentiation of imipenem against a resistant E. coli strain will serve as the basis for future in vivo studies.

Materials and Methods

General Procedure for Azetidinimine Synthesis. The imine (1.0 equiv.), the ynamide (2.0 equiv.) and the additive -silica gel (1.0 equiv.) or ZnOTf2 (10 mol%) -were successively added in a microwave sealable tube and placed under argon before the addition of t-BuOLi -solid or 2.2 M in solution in THF -(2.0 equiv.) followed by extra dry DMF (0.3M). The sealed tube was placed in a microwave apparatus for 1 h at 100 °C. The crude material was purified by flash chromatography on silica gel or with preparative TLC. See Supporting Information for detailed synthetic procedures and characterizations of the compounds.

In vitro β-lactamase inhibition assay. IC50 values were determined against the panel of purified carbapenemases: OXA-48, KPC-2 and NDM-1 by spectrophotometric assay, using ULTROSPEC 2000 UV spectrophotometer and the SWIFT II software (GE Healthcare, Velizy-Villacoublay, France). Compounds were dissolved in DMSO stock solutions at 10 mM; more dilute stocks were subsequently prepared as necessary by dissolving them also in DMSO. Assay conditions were as follows: 100 mM phosphate buffer, pH 7 (supplemented with 50 μM Zn 2+ when testing NDM-1, and with 50 mM NaHCO3 when testing OXA-48), 100 μM imipenem (Sigma-aldrich, Saint-Quentin Fallavier, France). The reaction was monitored at 297 nm, time course 600 seconds at 25°C with 3 min of incubation (compound/carbapenemase). Each inhibitor compound was assayed at seven different concentrations, in triplicate for calculating an error value with 95% confidence interval (ρ < 0.05). IC50 values were determined using the equation IC50 = ((1/0.5 x v0) -m)/q, where v0 is the rate of hydrolysis of the reporter substrate (v0 being the rate measured in the absence of inhibitor), q the y axis intercept and m the slope of the resulting linear regression. Percentage of inhibition were obtained with a concentration of 10 μM of compound. were built starting from 7aaa by manual editing using UCSF Chimera package. [START_REF] Pettersen | UCSF Chimera-A visualization system for exploratory research and analysis[END_REF] The structures of enantiomers were generated using an in-house script. Molecular docking was performed using the GOLD suite [START_REF] Verdonk | Improved protein-ligand docking using GOLD[END_REF] (CCDC) and the GoldScore scoring function, with the structures 4HL2, 44

Minimal

2OV5 [START_REF] Ke | Crystal Structure of KPC-2: Insights into Carbapenemase Activity in Class A β-Lactamases[END_REF] and 4S2P [START_REF] King | Molecular Mechanism of Avibactam-Mediated β-Lactamase Inhibition[END_REF] as receptors for NDM-1, KPC-2 and OXA-48, respectively. The binding sites were defined as 15 Å radius spheres centered on the Zn1 ion for NDM-1 and on the OG atom of Ser70 for KPC-2 and OXA-48. In agreement with our previous studies [START_REF] Surpateanu | Evaluation of docking performance in a blinded virtual screening of fragment-like trypsin inhibitors[END_REF][START_REF] Colas | Virtual screening of the SAMPL4 blinded HIV integrase inhibitors dataset[END_REF][START_REF] Martiny | Blind Pose Prediction, Scoring, and Affinity Ranking of the CSAR 2014 Dataset[END_REF][START_REF] Selwa | Molecular docking performance evaluated on the D3R Grand Challenge 2015 drug-like ligand datasets[END_REF][START_REF] Selwa | Blinded evaluation of farnesoid X receptor (FXR) ligands binding using molecular docking and free energy calculations[END_REF][START_REF] Chaput | Blinded evaluation of cathepsin S inhibitors from the D3RGC3 dataset using molecular docking and free energy calculations[END_REF][START_REF] Elisée | Performance evaluation of molecular docking and free energy calculations protocols using the D3R Grand Challenge 4 dataset[END_REF] showing that an enhanced conformational search is beneficial, especially for large molecules, a search efficiency of 200% was used to better explore the ligand conformational space. All other parameters were used with the default values. Images were generated using UCSF Chimera. [START_REF] Pettersen | UCSF Chimera-A visualization system for exploratory research and analysis[END_REF]

Figure 1 .

 1 Figure 1. Representative structures of (a) β-lactam antibiotics (in bold: year of entry to the

Scheme 1 .

 1 Scheme 1. (a) Staudinger synthesis to access β-lactams; (b) imino-Staudinger synthesis to access

Figure 2 .

 2 Figure 2. Docking of compound 7aaa in the active site of NDM-1 (left) and schematic drawing

Figure 4 :

 4 Figure 4: Docking conformations of the enantiomers R (a, b, c) and S (d, e, f) of compound 7dfm, in the active site of NDM-1 (class B) represented as gray surface (a, d), KPC-2 (class A) represented as light blue surface (b, e) and OXA-48 (class D) represented as pink surface (c, f). For NDM-1, the zinc ions are colored in purple, for KPC-2 the residues Ser70, Lys73 and Ser130 are colored in yellow, purple and brown, respectively, and for OXA-48 the residues Ser70 and Thr104 are colored in yellow and olive, respectively. Hydrogen bonds are represented as springs colored in orange.

  Inhibition concentrations. MIC values were determined by broth microdilution, in triplicate, in cation-adjusted Mueller Hinton broth according to the Clinical Laboratory Standards Institute (CLSI, https://clsi.org/) guidelines. The enterobacterial clinical strain E. coli NDM-1 GUE expressing the carbapenemase NDM-1 was used.[START_REF] Bonnin | Characterization of an IncFII Plasmid Encoding NDM-1 from Escherichia coli ST131[END_REF] Experiments were performed in microtiter plates containing the medium with imipenem and inhibitors (dissolved in DMSO).Three inhibitor concentrations were tested: 50, 100 and 200 μM. Plates were incubated overnight at 37°C for 18-24 h.Incubations in hepatic microsomes. Compounds (5 μM) were incubated in 0.5 mg/mL of pooled male mouse liver microsomes (from Biopredic, France), in 0.1 M phosphate buffer at pH 7.4 at 37 °C. After prewarming the mixture for 5 min, reactions were initiated by the addition of NADPH (1 mM). Incubations (400 µL) were performed at 37 °C for 0, 5, 15, 30 and 45 min in duplicate and the reaction immediately terminated by adding 200 μL of cold acetonitrile. Samples (including the control to evaluate non NADPH-dependent stability) were centrifuged and the supernatant fractions analyzed by UPLC-MS/MS with multiple reaction monitoring (MRM). Diphenhydramine was used as positive control in mouse liver microsomes test. The MRM area response of the analyte was set to 100% with the T0 incubation, the relative decrease in MRM area ratio intensity over time against that of the control (percent parent decrease) was used to determine the half-life (t1/2) of compounds in the incubation.Half-life values were calculated from the relationship: T1/2 (min) = 0.693/k, where k is the slope of the Ln concentration vs time curve. The intrinsic clearance (CLint) was calculated as: CLint = (0.693 x incubation volume (μL))/(t (min) x mg of microsomal protein).Cell culture and proliferation assay. Assays were carried out at the Institut de Chimie des Substances Naturelles by the CIBI screening platform. Cell lines were obtained from the American type Culture Collection (Rockville, USA) and were cultured according to the supplier's instructions. Briefly, human MRC-5 cells were grown in DMEM supplemented with 10% fetal calf serum (FCS) and 1% glutamine and HCT116 colorectal carcinoma cells were grown in RPMI 1640 containing 10% FCS and 1% glutamine. All cell lines were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Cell growth inhibition was determined by an MTS assay according to the manufacturer's instructions (Promega, Madison, WI, USA).Briefly, the cells were seeded in 96-well plates (2.5 × 103 cells/well) containing 200 μL of growth medium. After 24 h of culture, the cells were treated with the test compounds at different final concentrations. After 72 h of incubation, 40 μL of resazurin was added for 2 h before recording absorbance at 490 nm with a spectrophotometric plate reader. The IC50 value corresponded to the concentration of compound inducing a decrease of 50% in absorbance of drug-treated cells compared with untreated cells. Experiments were performed in triplicate. Paclitaxel was used as the reference compound. PCR, Cloning, Expression, and DNA Sequencing. Whole-cell DNA of the enterobacterales expressing β-lactamases NDM-1, NDM-4, NDM-7, NDM-9, VIM-1, VIM-52, CTX-M-15, CMY-2 and OXA-48 was extracted, using the QIAamp DNA mini kit (Qiagen, Courtaboeuf, France) and used as a template to amplify all the different genes. The sequences without the peptide signal (predicted by SignalIP 4.1 Server, http://www.cbs.dtu.dk/services/SignalP-4.1/) encoding for the mature protein, were obtained by PCR amplification, using the forward primers, which included an NdeI restriction site, and the reverse primer which included an XhoI restriction site and a deletion of the stop codon of the gene to allow the expression of an C_Term His tag.Then, PCR product was cloned into pET41b vector (Invitrogen ® , Life Technologies, Cergy-Pontoise, France), using NdeI and XhoI restriction enzymes, to obtain a C-Term His 8 -tag. The accuracy of the recombinant plasmid was verified by sequencing, using a T7 promoter and T7 terminator with an ABI Prism 3100 automated sequencer (Applied Biosystems, Thermo Fisher Scientific, Les Ulis, France). The nucleotide sequences were analyzed by using software available at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov).Protein purification. An overnight culture of E. coli BL21 DE3 harboring recombinant pET41b plasmids was used to inoculate 2 L of LB medium broth containing 50mg/L kanamycin. Bacteria were cultured at 37 °C until an OD of 0.6 at 600 nm was reached. The expression of the βlactamase genes was carried out overnight at 22 °C with 0.2 mM IPTG as inducer. Cultures were centrifuged at 6000 g for 15 min and then the pellets were resuspended with the binding buffer (10 mM imidazole, 25 mM sodium phosphate pH 7.4 and 300 mM NaCl). Bacterial cells were disrupted by sonication and the bacterial pellet was removed by two consecutive centrifugation steps at 10000g for 1h at 4 °C; the supernatant was then centrifuged at 96000g for 1h at 4 C. The soluble fractions were filtered and then passed through a HisTrapTM HP column (GE Healthcare) and proteins were eluted with the elution buffer (500 mM imidazole, 25 mM sodium phosphate pH 7.4 and 300 mM NaCl). Finally, a gel filtration step was performed with 100 mM sodium phosphate buffer pH 7 and 150 mM NaCl with a Superdex 75 column (GE Healthcare). The protein purity was estimated by SDS-PAGE. The pooled fractions were dialyzed against 10 mM Tris-HCl pH 7.6, for NDM and VIM 50 µM of ZnSO4 was added, then concentrated using Vivaspin columns (Sartorius, Aubagne, France). The concentrations were determined by measuring the OD at 280 nm and with the extinction coefficients obtained from the ProtParam tool (Swiss Institute of Bioinformatics online resource portal).[START_REF] Artimo | ExPASy: SIB bioinformatics resource portal[END_REF] Molecular modeling. The three-dimensional structure of compound 7aaa[START_REF] Romero | Base-Mediated Generation of Ketenimines from Ynamides: Direct Access to Azetidinimines by an Imino-Staudinger Synthesis[END_REF] was retrieved from the Cambridge Structural Database 42 (CSD refcode KEMJEU) and that of compound 7dfm

  Selwa, P. Retailleau and B. I. Iorga. K. Cariou and B. I. Iorga drafted the manuscript which was amended and commented on by all authors. # E. R. & S. O contributed equally; ◊ M. B. & A. C. A. D. contributed equally . Conflict of Interest Disclosure: The authors declare no competing financial interest List of Abbreviations CTX M 15: Cefotaximase-Munich 15 CMY-2: Cephamycinase-2 DMSO: dimethyl sulfoxideGNB: Gram-negative Bacilli HCT 116: Human Colorectal Carcinoma cells KPC: Klebsiella Pneumoniae Carbapenemase MBL: Metallo-β-lactamase MIC: Minimum Inhibitory Concentrations MRC-5: Medical Research Council cell strain 5 ND: Not Determined NDM: New Delhi metallo-β-lactamase NE: no effect OXA: Oxacillinase SAR: Structure Activity Relationship SBL: Serine-β-lactamases

Table 1 .

 1 Influence of Ar 3 on NDM-1, OXA-48 and KPC-2 inhibitory activities compared to 7aaa.

	IC50 (µM) or % inhibition

N.E.: no effect (at 10 µM); N. D.: not determined

Table 2 .

 2 Influence of Ar 2 on NDM-1, OXA-48 and KPC-2 inhibitory activities compared to 7aaa.

	IC50 (µM) or % inhibition

Table 3 .

 3 Influence of Ar 1 on NDM-1, OXA-48 and KPC-2 inhibitory activities compared to 7aba and 7cfa.

	IC50 (µM) or % inhibition

N.E.: no effect (at 10 µM)

Table 4 .

 4 Influence of Ar 1 on NDM-1, OXA-48 and KPC-2 inhibitory activities compared to 7aha and 7ahb.

	IC50 (µM) or % inhibition

Table 5 .

 5 Influence of Ar 2 compared to 7dba and of Ar 3 compared to 7dfa on NDM-1, OXA-48 and KPC-2 inhibitory activities

	IC50 (µM)

then performed complementary assays to ascertain its therapeutic potential. First, it was screened at 10 µM against a wider panel of BLs (Table

6

). At this concentration a complete inhibition of three NDM variants (NDM-4, NDM-7 and NDM-9) and of VIM-1 (Class B) was observed. In contrast, VIM-52 (class B) was not affected. The extended-spectrum β-lactamase CTX-M-15 (class A) and the cephalosporinase CMY-2 (class C) were also inhibited (83% and 86%, at 10 µM, respectively). These latter results demonstrate that compound 7dfm can inhibit not only carbapenemases from 3 classes but, more generally, BLs from all four classes.

Table 6 .

 6 Additional enzymatic inhibitory activities (% at 10 µM) for 7dfm

	Compound	NDM-4	NDM-7 NDM-9	VIM-1	VIM-52	CTX-M-15	CMY-2
	7dfm	100%	100%	100%	100%	0%	83%	86%

Table 7 .

 7 Metabolic stability, cytotoxicity and imipenem repotentiation for 7dfm Stability after 45 min in the presence of mouse hepatic microsome in the absence of NADPH; b CLint was determined using mouse hepatic microsomes and is given in µL/min/mg protein; c inhibition of cell proliferation; d Minimum inhibitory concentration for imipenem using E. coli GUE-NDM1 39 clinical strain.

		Stability	IC50 (µM) c		MIC (µg/mL) d
		Non-NADPH a	CLint b	MRC-5	HCT-116	0µM	20µM	50µM
	7dfm	96%	13.9	19.9	32.3	16	8	4

a

Acknowledgments

This work was supported by the Laboratory of Excellence in Research on Medication and Innovative Therapeutics (LERMIT, grant ANR-10-LABX-33 under the program Investissements d'Avenir ANR-11-IDEX-0003-01), the FCS Campus Paris-Saclay pre-maturation program (project Inhibase), the SATT Paris-Saclay (project CARBAMAT), the JPIAMR transnational project DesInMBL (grant ANR-14-JAMR-0002) and the Région Ile-de-France (DIM Malinf). The authors also thank CNRS, AP-HP, Université Paris-Saclay, and ICSN, for financial support.

Supporting Information

The Supporting Information contains detailed synthetic procedures and characterizations of the compounds and copies of the NMR spectra.

Author Information Author Contributions