O Galangau

D Daou

N El Beyrouti

E Caytan

C Meriadec

F Artzner

S Rigaut

Molecular Engineering onto Ru II bis(1,2-diphenylphosphinoethane) synthon: toward a new family of organometallic gelator

In this article we report the successful molecular engineering of Ru bis-acetylides that led for the first time to gelators, and more specifically in aromatic solvents. By means of a non-linear ligand and an extended aromatic platform, the bulky Ru bisacetylides were able to self-assemble into lamellar structures as evidenced by SEM in benzene, toluene, o-and m-xylene, which in turn induced gelation of the solution with critical gelation concentration of 30 mg / mL. NMR, VT-NMR, FT-IR spectroscopies evidenced that hydrogen bonds are mainly responsible for the self-organization. VT-NMR and SAXS have also suggested that the pro-ligand and the complex stack in different ways.

For Table of Content only

A Ru II bis-acetylide complex is able to form organic gels for the first time, thanks to a dedicated molecular design.

Gel

Ru 1 Ru 2   No Gel

INTRODUCTION

Supramolecular interactions are key features to obtain (multi-) functional materials with applications ranging from optoelectronics [START_REF] Amabilino | Supramolecular materials[END_REF][START_REF] Li | Molecular Packing: Another Key Point for the Performance of Organic and Polymeric Optoelectronic Materials[END_REF] to molecular electronics. [START_REF] Hasegawa | Conducting supramolecular nanofibers and nanorods[END_REF][START_REF] Wang | Organic field-effect transistors based on highly ordered single polymer fibers[END_REF][START_REF] Faramarzi | Lighttriggered self-construction of supramolecular organic nanowires as metallic interconnects[END_REF] To that regard, molecular gels [START_REF] Weiss | The past, present, and future of molecular gels. What is the status of the field, and where is it going?[END_REF] have received a great deal of attention in the last decade for their appealing properties which have permitted developments of materials with high chemical sensing abilities, [START_REF] Kartha | Attogram sensing of trinitrotoluene with a self-assembled molecular gelator[END_REF][START_REF] Babu | Functional pigelators and their applications[END_REF][START_REF] Babu | Selfassembled gelators for organic electronics[END_REF][START_REF] Yang | Stimuli responsive gels based on low molecular weight gelators[END_REF] remarkable charge transport properties, [START_REF] Zhang | Supramolecular Self-Assembly in a Submicrometer Electrodic Cavity: Fabrication of Heat-Reversible pi-Gel Memristor[END_REF] photochemical and electrochemical responsiveness. [START_REF] Yang | Stimuli responsive gels based on low molecular weight gelators[END_REF][START_REF] Draper | Photoresponsive gelators[END_REF][START_REF] Sun | Multistimuli-responsive supramolecular gels: design rationale, recent advances, and perspectives[END_REF][START_REF] Yan | Stimuli-responsive supramolecular polymeric materials[END_REF] Gels can be defined as soft materials made of a liquid phase (major component) immobilized in a 3D cross-linked network (minor component). This network can be made of covalent polymeric chains or prepared from the supramolecular assembly of a low molecular weight compound, both being known as the gelator. For the so-called supramolecular gels, the gelator self-assembles thanks to a combination of weak interaction such as Van der Waals or electrostatic interactions, hydrogen/halogen bonding and/or - stacking interactions. A gel is commonly identified by the test inversion method which consists in looking at the ability of the soft material to support its own weight when the vial is turned upside-down. Therefore, a gel can be interpreted as a clear sign of self-assembly properties whereas the lack of gelation may stem from a limited propensity to form supramolecular polymers. Recently, metallogels [START_REF] Zhang | Metal-organic gels: From discrete metallogelators to coordination polymers[END_REF][START_REF] Fages | Metal coordination to assist molecular gelation[END_REF][START_REF] Tam | Recent advances in metallogels[END_REF][START_REF] Dastidar | Metallogels from Coordination Complexes, Organometallic, and Coordination Polymers[END_REF] where the gelator is either a coordination or an organometallic derivative have drawn a lot of attention. Indeed, the presence of the metal has brought about new features such as redox responses, [START_REF] Gasnier | Metallo-supramolecular gels based on a multitopic cyclam bis-terpyridine platform[END_REF] luminescence [START_REF] Kotova | Europium-directed self-assembly of a luminescent supramolecular gel from a tripodal terpyridine-based ligand[END_REF] or catalytic activities. [START_REF] Beatriu Escuder | Supramolecular gels as active media for organic reactions and catalysis[END_REF] They have also found applications in waveguiding [START_REF] Wang | Cooperative Supramolecular Polymerization of Fluorescent Platinum Acetylides for Optical Waveguide Applications[END_REF] or as anticancer treatments. [START_REF] Zhang | III) supramolecular polymers for anticancer treatment[END_REF] Among all metallogelators, rigid rod-like metal-acetylides, where the metal is Pt(II) 24- 27 or Au(III), [START_REF] Au | Luminescent metallogels of bis-cyclometalated alkynylgold(III) complexes[END_REF] are efficient gelators thanks to their flat structures, and for Pt(II) centers, their ability to provide additional Metal-Metal interactions [START_REF] Tam | Luminescent metallogels of platinum(II) terpyridyl complexes: interplay of metal...metal, pi-pi and hydrophobic-hydrophobic interactions on gel formation[END_REF][START_REF] Camerel | Formation of gels and liquid crystals induced by PtPt and pi-pi* interactions in luminescent sigma-alkynyl platinum(II) terpyridine complexes[END_REF] that are further driving the supramolecular assembly. Because of their photophysical properties, those metal-acetylides metallogels were mainly use as light emitting soft materials. Alternatively, metallogels transporting electrical charges are well desired for the development of flexible, lightweight, electronic devices, although examples were scarcely documented so far. [START_REF] Wu | Availability, Applicability, and Advanceability[END_REF] To obtain rigid-rod conductive supramolecular metallogels, organometallic acetylides gelators must combine strong supramolecular abilities to low potential and reversible redox processes.

Chart 1. Molecular structures of Ru1 and Ru2 complexes

Our group, among others, has been involved in the chemistry of mono-and bimetallic Ru(II) bis-acetylide complexes holding great promises for optoelectronic applications. Indeed, these compounds feature remarkable charge transport properties in nanojunctions [START_REF] Luo | Length and Temperature Dependent Conduction of Ruthenium-Containing Redox-Active Molecular Wires[END_REF][START_REF] Meng | Orthogonally modulated molecular transport junctions for resettable electronic logic gates[END_REF][START_REF] Schwarz | Field-induced conductance switching by charge-state alternation in organometallic single-molecule junctions[END_REF][START_REF] Tanaka | Single-molecule junctions of multinuclear organometallic wires: long-range carrier transport brought about by metal-metal interaction[END_REF][START_REF] Milan | Metal bis(acetylide) complex molecular wires: concepts and design strategies[END_REF][START_REF] Naher | Structure and Physical Properties of "Wire-like" Metal Complexes[END_REF][START_REF] Zhang | Ruthenium(II) as Conductive Promoter To Alleviate Conductance Attenuation in Oligoynyl Chains[END_REF][START_REF] Wuttke | Fully delocalized (ethynyl)(vinyl)phenylene bridged triruthenium complexes in up to five different oxidation states[END_REF][START_REF] Ou | Bonding and Electronic Properties of Linear Diethynyl Oligothienoacene-Bridged Diruthenium Complexes and Their Oxidized Forms[END_REF][START_REF] Ying | Linear trimer of diruthenium linked by butadiyn-diyl units: a unique electronic wire[END_REF] , on gold surfaces as charges storage layer [START_REF] Mulas | Fast Electron Transfer Exchange at Self-Assembled Monolayers of Organometallic Ruthenium(II) sigma-Arylacetylide Complexes[END_REF] or as "push-pull" chromophoric reagents for solar cells [START_REF] Colombo | A Novel Diruthenium Acetylide Donor Complex as an Unusual Active Material for Bulk Heterojunction Solar Cells[END_REF] and NLO applications. [START_REF] Roberts | Organometallic Complexes for Nonlinear Optics. 45. Dispersion of the Third-Order Nonlinear Optical Properties of Triphenylamine-Cored Alkynylruthenium Dendrimers[END_REF][START_REF] Simpson | Record Multiphoton Absorption Cross-Sections by Dendrimer Organometalation[END_REF][START_REF] Samoc | Two-photon and three-photon absorption in an organometallic dendrimer[END_REF][START_REF] Zhang | Nonlinear optical properties of meso-Tetra(fluorenyl)porphyrins peripherally functionalized with one to four ruthenium alkynyl substituents[END_REF][START_REF] Triadon | Linear and Third-Order Nonlinear Optical Properties of Fe(η5-C5Me5)(κ2-dppe)and trans-Ru(κ2-dppe)2-Alkynyl Complexes Containing 2-Fluorenyl End Groups[END_REF] Recently, we also reported Ru bis-acetylides as efficient electrochemical switch for lanthanide emission in the NIR ranges [START_REF] Norel | Lanthanide Sensitization with Ruthenium Carbon-Rich Complexes and Redox Commutation of Near-IR Luminescence[END_REF][START_REF] Sabea | Dual Light and Redox Control of NIR Luminescence with Complementary Photochromic and Organometallic Antennae[END_REF] or magnetic properties. [START_REF] Norel | A carbon-rich ruthenium decorated dysprosium single molecule magnet[END_REF] However, to the best of our knowledge, there is no example of mono-or bimetallic Ru(II) bis-acetylide derivatives able to form supramolecular assemblies / gels, that would widen their application field, thanks to their unique electronic properties. This statement presumably result from the fact that the stabilizing 1,2-dppe ancillary ligands prevent accurate intermolecular contacts. [START_REF] Vives | Synthesis of a nanocar with organometallic wheels[END_REF] Therefore, designing Ru(II) bis-acetylide complexes able to overcome this drawback and form supramolecular architectures accommodating its bulky ligands is challenging. We anticipated that placing an acetylide ligand, featuring a simple hydrogen donating/accepting moiety in trans position with respect to the metal center, would not be sufficient for driving self-assembling, as it is for Pt(II) bis-acetylides [START_REF] Wang | Cooperative Supramolecular Polymerization of Fluorescent Platinum Acetylides for Optical Waveguide Applications[END_REF] derivatives. Therefore, we selected a ligand that could trigger gelation of alkane solvents, while connected to a bulky coordination sphere, such as those used in the Ln(III) complexes developed by Ziessel, Camerel, Charbonnière. [START_REF] Kadjane | Divergent approach to a large variety of versatile luminescent lanthanide complexes[END_REF][START_REF] Camerel | Tuning the thermotropic and lyotropic properties of liquid-crystalline terpyridine ligands[END_REF] This ligand encompasses a central toluyl fragment connected to two amide functions that, on the basis of crystallographic data, was found to pack in a head-to-tail fashion with a molecular packing driven by hydrogen bonding interactions. The toluyl fragment is essential since it prevents the amide functions to planarize (see Supporting Information). This article will disclose the synthesis, the physicochemical properties of two original symmetrical monometallic Ru(II) bisacetylide complexes featuring this efficient platform (Chart 1). It was also found that Ru2 which encompasses an extended ligand aiming at further separating the hydrogen bonding sites from the 1,2-dppe crowded unit and favoring additional - stacking interactions, and hence remotely positioned hydrogen bonding sites to afford organogels in aromatic solvents.

RESULTS AND DISCUSSIONS

Syntheses. Ru1 and Ru2 derivatives were obtained from a multistep synthetic procedure in very good yields (Scheme 1). Both compounds are air-and bench stable yellow powders. The starting compound A was converted into compound 1 through Sonogashira coupling (97%). Aniline functional groups of 1 were further coupled to the gallic acid C presenting three dodecyl alkyl chains, with mild conditions, to afford the intermediate derivative 2 (91%). Finally, deprotection of the TMS group of 2 afforded L 1 quantitatively. A similar synthetic path was chosen for preparing L [START_REF] Li | Molecular Packing: Another Key Point for the Performance of Organic and Polymeric Optoelectronic Materials[END_REF] . The difference lies in the first step. Compound A was coupled to derivative B featuring a TMS-protected phenyl acetylene moiety to yield intermediate 3 in a quantitative manner. Note that L 1 and L 2 are stable for months, at room temperature and under air. Finally, Ru1 and Ru2 complexes were obtained in 83% and 82% yields, respectively, in presence of NaPF6 / Et3N. [START_REF] Olivier | Redox-Active Molecular Wires Incorporating Ruthenium(II) σ-Arylacetylide Complexes for Molecular Electronics[END_REF] The temperature was maintained at 30°C to ensure proper solubility of the pro-ligands. These Ru complexes were characterized with 1 H, [START_REF] Sun | Multistimuli-responsive supramolecular gels: design rationale, recent advances, and perspectives[END_REF] C, [START_REF] Wu | Availability, Applicability, and Advanceability[END_REF] P, FT-IR spectroscopies and HR-MS, and a full set of complementary characterizations can be found in the supporting information (spectrophotometric features). We have also probed their electrochemical behavior. Both compounds undergo a fully reversible one electron oxidation process at around 0.4 V vs SCE in DCM (0.2 M n Bu4NPF6) at room temperature as we expected (supporting information). Finally, we also prepared compound 5, which does not present the Ru-centered building block, in order to investigate the importance of the molecule rod-like shape. As a result, derivative 5 was obtained from the homocoupling of L 2 in Glaser conditions in poor yields (17%). This low yield probably originates from the very poor solubility of 5 in common organic solvents which prevent high yield purification. Gelation properties. The gelation properties of L 2 , Ru1, Ru2 and 5 studied by the inversion method are summarized in Table 1. The pro-ligand L 2 was able to form transparent gels (Figure 1) in alkane solvents such as cyclohexane, methylcyclohexane, n-dodecane or n-heptane with CGC (Critical Gelation Concentration) of 1.3 mg / mL, except for methylcyclohexane (4.0 mg / mL). These values are similarly to those found for other organic gelators possessing an analogous structure. [START_REF] Camerel | Tuning the thermotropic and lyotropic properties of liquid-crystalline terpyridine ligands[END_REF] As previously reported by Ziessel et al., 56 L 1 affords turbid gels in acetone only. Therefore, extending the aromatic platform with L 2 has markedly improved the overall gelation ability. Interestingly, gelation abilities are annihilated with dimer compound 5 as it is not soluble in any solvents used in this study, except in CHCl3 which was used to characterize it. Concerning the complexes, Ru1 does not form any gels but precipitates in cyclohexane and methylcyclohexane when cooling down to room temperature. In aromatic solvents, the molecule is still soluble after cooling. On the contrary, Ru2 derivative affords turbid gels in aromatic solvent, while in alkanes the compound is not soluble. CGCs of approximatively 30 mg / mL were obtained for benzene, o-and m-xylene, while a CGC of 20 mg / mL was measured for toluene. When compared to best organic gelator found in the literature (CGC < 1 mg / mL) Ru2 gelation aptitudes are moderate but comparable to others acetylide complexes. For example, in 2011, Yang's group reported a series of bimetallic Pt(II) bis-acetylides complexes featuring an iptycene as central bulky fragment. [START_REF] Zhang | Platinum Acetylide Complexes Containing Iptycene as Cores: A New Family of Unexpected Efficient Organometallic Gelators[END_REF] Unexpectedly, these molecules formed gels in alkanes with CGC ranging from 20 to 65 mg / mL, However, in the present case Ru2 provides the first example of its kind accommodating highly sterically demanding ancillary ligand. Furthermore, comparing compounds 5 and Ru2 indicates that the dppe ligands play a key role in solubilizing and presumably in forming gels in aromatic solvents via probable - stacking interactions. We have also measured the gel-to-solution transition temperatures by immersing the samples in a temperaturecontrolled bath. Transition temperature refers to the temperature at which the gel (at the CGC) starts to melt and collapses when the tube is turned upside down. Regarding pro-ligand L 2 , transition temperatures of 55°C were found in n-heptane and ndodecane, whereas values of 43°C and 45°C were obtained in cyclohexane and methylcyclohexane, respectively. As a result, gels of L 2 in linear alkanes are more robust. Gels of Ru2 in aromatic solvents display transition temperatures of 24°C in toluene, 31°C in o-and m-xylene and 34°C in benzene. Gels of Ru2 and of L 2 are stable for at least two months in usual laboratory conditions. Even after this period of time, the gelation process can be repeated at least 5-10 times, with no loss of apparent efficiency. Note that these gels do not show any thixotropic behavior. In short, comparison between derivatives L 2 , 5, Ru1 and Ru2 clearly demonstrates the importance of i) the 1,2-dppe ligand which brings about some solubility and gelling ability in aromatic solvents, ii) extending the aromatic surface to favor self-assembly probably overcome the steric hindrance provided by the the dppe crown leading to more available hydrogen bonding sites, and extending the aromatic platform to offer more possibilities for the molecules to pack thanks to - stacking interactions in particular in L 2 (vide infra). Comparing Ru1 and Ru2 1 H NMR spectra in CD2Cl2 where no supramolecular interactions take place (see supporting information), we observed that, at room temperature, the signal assigned to aromatic protons located on the toluyl fragment experiences a drastic downfield shift from Ru1 to Ru2 (from 6.80 ppm to 7.70 ppm) while the hydrogen atoms located on the gallate moieties are not affected. This shift evidences the magnetic influence of the dppe crown on these protons. Moreover, signals attributed to the methyl and the amide protons are also similarly influenced by the dppe to a minor extent. Therefore, it is reasonable to think that in Ru1 supramolecular packing via hydrogen bonding and possible - stacking is prevented because of the bulkiness of the ancillary ligand, which in turn explains why it does not afford gels in aromatic solvent when Ru2 does (vide infra). This result sheds light on the importance of the ligand design and on the importance of the dppe crowding. 20) G ( 30) G (33) Numbers in bracket represent the Critical Gelation Concentration given in mg / mL. G = Gel, P = Precipitate after cooling down, NS = Not Soluble and S = Soluble after cooling down (no gel).

To further support this statement, we have transposed to our complexes the model of Cavallo et al., originally developed for a better understanding of catalytic activities of organometallic complexes. [START_REF] Falivene | Towards the online computer-aided design of catalytic pockets[END_REF] Cavallo's calculations provide i) the percentage of buried volumes, i.e. the space occupied by a ligand in the first coordination sphere of a metal center or a putative atom, and ii) the corresponding topographic steric map of a ligand. Consequently, this map provides a quantitative description of the interaction surface between the sphere center and a substrate. Assuming that the approach of a "substrate" could be regarded as a supramolecular overlap, calculating the buried volume (steric map) at a judiciously chosen point along the molecular axis is highly relevant to apprehend the steric bulk of the dppe units and the possibilities of interactions in the present case. To achieve these calculations we first simulated Ru1 and Ru2 geometries with a DFT method (see supporting information for more details).Then, we used these results to calculate the Cavallo's parameters for a putative atom located at the center of the toluyl fragment along the Ru1 and Ru2 long molecular axis. In Ru2, the percentage of buried volume (%Vbur) is calculated to be 0% reflecting a free environment available for supramolecular interactions, while in Ru1, at the same location, %Vbur is found to be 23%, indicating that the dppe chelate probably precludes ligand overlapping and further supramolecular interactions. In the next sections, we now describe our further studies on the gels and xerogels morphologies thanks to Scanning Electron Microscopy and FT-IR.

Scanning Electron Microscopy and FTIR Studies (SEM).

SEM was performed to study the morphology of the corresponding xerogels (freeze-dried gels) of compounds L 2 and Ru2. As observed in Figure 2, a 3D network comprising entangled fibers was obtained for xerogel of L 2 , with fibers diameters of 100 -300 nm. In contrast, Ru2 presents lamellar objects with width of approximatively 200 -400 nm, along with spherical particles with diameters ranging from 200 to 1000 nm. The detection of two different type of supramolecular objects remains elusive so far. Spherical particles might be due to self-aggregation during the freeze-drying step. Nevertheless, these SEM experiments proves the capability of Ru2 compound to self-assemble into supramolecular ribbons. FT-IR spectrum of these xerogels displays the vibration of the amide C=O bond at 1635 cm -1 for L 2 and 1637 cm -1 for Ru2 (Figure 3). In addition, the N-H vibration from the amido moieties was found at 3153 cm -1 for L 2 and 3230 cm -1 for Ru2. These values are characteristic of hydrogen-bonded molecules and are close to values found for very similar molecular skeleton. [START_REF] Camerel | Tuning the thermotropic and lyotropic properties of liquid-crystalline terpyridine ligands[END_REF] .

NMR studies of gelation.

To get more insight into the supramolecular processes, the gel formation was monitored by 1 H Variable Temperature NMR (VT-NMR) studies in C6D12 for L [START_REF] Li | Molecular Packing: Another Key Point for the Performance of Organic and Polymeric Optoelectronic Materials[END_REF] and C6D6 for Ru2. Starting with compound L 2 (see supporting information), the broad signal located at 8.3 ppm assigned to amide groups protons experiences an downfield shift (+0.61 ppm) as the temperature decreases from 340 K to 320 K. This is a clear evidence of the involvement of the hydrogen bonds into the supramolecular process as indicated in the xerogel with IR spectroscopy. Interestingly, aromatic signals experience an upfield shift as well as the methyl protons signal located 2.00 ppm (-0.13 ppm). Finally, the alkynyl proton exhibits no shift which leads us to think that this part of the molecule is not involved in the supramolecular packing. Additional 2D ROESY experiment performed at 325 K features a cross-correlation between the alkynyl proton and some protons from the aliphatic chains (Figure S29), which proves that these two group of atoms are spatially close during the self-assembly process. These observations are consistent with a head-to-tail supramolecular assembly as previously suggested by Ziessel et al on an analo-

a) b) c) d)
gous system (see supporting information). [START_REF] Camerel | Tuning the thermotropic and lyotropic properties of liquid-crystalline terpyridine ligands[END_REF] As far as Ru2 derivative is concerned, we have conducted 1 H and 31 P VT-NMR experiments in C6D6, at the CGC (Scheme 3). First, [START_REF] Wu | Availability, Applicability, and Advanceability[END_REF] P{ 1 H} experiment displays one singlet signal located at approx. 53.5 ppm at 340 K, a value which is characteristic of a Ru(II) trans-bisacetylide complex. By slowly cooling down the sample to 295 K, the singlet signal undergoes a slight shift and vanishes because of gelation at 300 K. This singlet signal reappears when the sample is warmed above the gelation temperature. We repeated this experiment at least four times and showed great reproducibility with no obvious degradation. Therefore, we can safely conclude that Ru2 is chemically stable in that temperature range, as already suggested by qualitative observations. Regarding the 1 H NMR spectrum, the broad singlet signal located at 7.45 ppm and assigned to the amide protons experiences a significant downfield shift from 340 K to 300 K (+0.55 ppm). This is in line with molecules interacting trough hydrogen bonds, presumably between their amide groups, as for L [START_REF] Li | Molecular Packing: Another Key Point for the Performance of Organic and Polymeric Optoelectronic Materials[END_REF] . Importantly, the singlet signal located at 2.20 ppm (methyl protons) also experiences a downfield shift. This is in stark contrast with what was observed on ligand L 2 and might suggest, at this stage, that Ru2 does not pack the same way as L 2 does. However, the choice of solvent deeply matters in the context of supramolecular chemistry and therefore, the comparison between L 2 and Ru2 may not be so straightforward. [START_REF] Mabesoone | Solute-Solvent Interactions in Modern Physical Organic Chemistry: Supramolecular Polymers as a Muse[END_REF] Interestingly, multiplet signals due to the dppe aromatic protons does not shift with temperature indicating that the dppe is not involved in the supramolecular process, as we anticipated. Additional 2D NOESY and 2D ROESY performed at 315 K exhibit cross-correlations indicating several dipolar coupling between protons of the toluyl fragment, the amido groups and the fatty chains (Figure S30 & S31). These through-space couplings support the existence of supramolecular contacts involving the acetylide ligands. Further variable temperature DOSY-NMR (Figure S23) were conducted in C6D6 at the CGC to measure the gel-to-solution transition temperature. The molecular diffusion coefficient reached a plateau at 32°C, meaning that the gel state was obtained. This value is in good agreement with the one measured previously (Figure 1). To sum up, FT-IR combined to NMR and VT-NMR experiments have clearly established that hydrogen bonds are involved in the construction of the supramolecular assemblies. On one hand L 2 seems to behave as reported by Ziessel et al., with molecules packing in a head-to-tail fashion and rejecting the alkynyl protons out of the structure. On the other hand, whereas Ru2 compound also exhibits hydrogen bonding that are central for the assembly (downfield shift of the amide proton signal), the methyl signal also experiences a downfield shift which is inconsistent with a head-to-tail supramolecular packing. Eventually, VT-NMR and 2D NOESY and 2D ROESY clearly evidences that dppe are not involved in the supramolecular process.

Small Angle X-ray Scattering (SAXS).

To get a better insights into the supramolecular architectures of both gels, we performed Small Angle X-ray Scattering experiments. The X-ray scattering of the ligand L 2 (Figure 4) exhibits two peaks at 0.344Å -1 and 0.69Å -1 corresponding to long-range order with a lamellar repeat distance aL of 18.2Å (supporting information, Figure S33). An additional broad scattering is observed at 0.15Å -1 indicating a short-range order interdistance bL of 42±5Å. This suggests a lamello-columnar organization. In this hypothesis, the columnar cross section is SL = aL bL = 760 Å 2 . These arrangements and surface are close to those of the parent system et al [START_REF] Camerel | Tuning the thermotropic and lyotropic properties of liquid-crystalline terpyridine ligands[END_REF] and confirms the column formation tendency observed by NMR and FTIR. Note that the orientation of the diamido backbone can be either in plane or perpendicular to the layer. However this can be clarified by noting that the aromatic volume ratio is between 25% and 30%. This imposes the mean thickness of the aromatic part in the lamellar structure to be thinner than 5.5Å which is incompatible with a perpendicular orientation. The in plane orientation is compatible with observed H-bonds and the head-to-tail packing hypothesis. Thus, the hierarchical structure of the gel is consequently formed by large fibers which are made of columns with a head-to-tail packing organized in layers by means of chain interdigitation. The X-ray scattering of Ru2 (Figure 4) exhibits a well-defined set of lamellar peaks at 0.158, 0.32, 0.480 and 0.65Å -1 corresponding to a repeat distance aRu of 39.2Å. The length of the aromatic-ethenyl-Ru rigid part is about 33Å. This is compatible with a perpendicular orientation to the layers. Interestingly, the amide backbone is parallel to the layer and therefore perpendicular to the mean plane containing the acetylide scaffold. In other words, Ru2 forms lateral intermolecular hydrogen bonds leading to layers of molecules showing a segregation between the aliphatic part and the aromatic part. Thanks to chain interdigitations, the layers assemble together affording large ribbons (Figure S33) responsible for the gelation. In particular, aromatic solvent molecules might be confined within the layers, therefore contributing to the overall solvent immobilization. Such a result echoes with the fact that the Ru2 gels are weak, fragile and feature moderate gel-to-sol transition temperatures. Overall, SAXS confirm that L 2 and Ru2 present different packing as already suggested by the VT-NMR experiments.

CONCLUSIONS

To conclude, we have successfully synthetized the first example of a Ru(II) bis-acetylide complex showing supramolecular capabilities. By means of a rational molecular engineering which consists i) in placing hydrogen bonding accepting/donating groups out of the molecule's long axis and ii) in extending the ligand platform in order to decrease the steric influence of the dppe crown, this complex was able to form ribbons in aromatic solvents, which in turn induced gelation of the solution. In particular, NMR, VT-NMR, FT-IR spectroscopies have evidenced that hydrogen bonds are mainly responsible for the self-organization. VT-NMR and SAXS have also suggested that both the pro-ligand and the complex self-assemble thanks to hydrogen bonding interactions. While L 2 packs in a head-to-tail fashion, Ru2 forms lateral hydrogen bonding with a segregation between the aliphatic and the aromatic parts. We are currently investigating the self-assembly mechanism, and the necessity for the complex to be symmetrical to generate supramolecular packing. We believe these results open the door to new opportunities for redox active Ru bis-acetylides compounds, and will encourage the development of new parent complexes featuring improved gelation efficiencies.

EXPERIMENTAL SECTION

General information. Chemicals and solvents (HPLC, spectroscopic grade) were purchased from Merck-Sigma Aldrich, Acros Organics, Alfa Aesar, Fisher Scientific and were used without any further treatment. Anhydrous (HPLC) solvents were obtained from a MBraun SPS-800 drying system. Dry THF was obtained after distillation on Na/Benzophenone. Dry Diisopropylamine (DIPA), dry Triethylamine (TEA) and dry DMF were obtained from after distillation on CaH2 and kept in the dark on molecular sieves. All reactions were carried out under argon atmosphere using Schlenk techniques, unless otherwise stated. Schlenk tubes were dried under vacuum using heat-gun. TLC analyses were achieved on silica gel and bought from Fluka (silica gel matrix containing a fluorescent indicator at 254 nm). Column chromatography were performed on silica gel from Acros Organics (pore size of 60 Å, particle size of 40-63 µm) unless otherwise stated. 1 H, [START_REF] Sun | Multistimuli-responsive supramolecular gels: design rationale, recent advances, and perspectives[END_REF] C and 31 P-NMR spectra were recorded on a Bruker Avance III 400 MHz, Bruker AMX-3 300 (300 MHz) at 303K. Relevant compounds were also characterized by 2D NMR using HSQC/HMQC, COSY 1 H-1 H, HMBC sequences. VT-NMR were recorded on a Bruker Ascend 500 MHz Av III HD. Deuterated solvents were bought from Euriso-top. Infrared spectra (KBr) were acquired on a spectrophotometer FTIR BRUKER EQUINOX 55. HR-MS spectra were obtained from the Centre Régional de Mesures Physiques de l'Ouest (CRMPO). Intermediate A was obtained through an adapted procedure from the literature (see supporting information). Precursors B and C were synthetized as reported in the literature (see supporting information for a reference). Electrochemical measurements. Electrochemical studies were carried out under argon using an Eco Chemie Autolab PGSTAT 30 potentiostat (CH2Cl2, 0.2M Bu4NPF6), the working electrode was a Carbon disk and decamethylferrocene the internal reference. Scanning Electron Microscopy (SEM). Metallization by Au/Pd. Scanning Electron Microscopy (SEM) of xerogels were evaluated using the JEOL IT 300 Scanning Electron Microscope. Samples were collected and deposited on a Teflon plot. Each sample was examined using a voltage of 5 or 10 kV. Images were analyzed by SMileView software.

Small Angle X-Ray Scattering (SAXS). Organogel and xerogel samples were prepared into capillaries. SAXS experiments were performed using X-ray patterns collected with a Pilatus 300k (Dectris, Grenoble, France) mounted on a microsource X-ray generator GeniX 3D (Xenocs, Sassenage, France) operating at 30 watts. The monochromatics CuKαradiation is of λ = 1.541 Å. The diffraction patterns were therefore recorded for reciprocal spacing q = 4π*sinθ/λ in a range of repetitive distances from 0.015 Å-1 (418 Å) and 1.77 Å-1 (8 Å). Images were transformed to graphics using the software program Fit2D (ESRF).

Syntheses. Compound 1.

A dried Schlenk tube was charged with compound A (0.200 g, 0.8 mmol, 1 eq.), PdCl2(PPh3)2 (0.085 g, 5 mol%) and CuI (0.023 g, 5 mol%) under argon flow. Then, distillated Et3N (12 mL) was added followed by dry THF (12 mL). To this mixture was added excess of neat TMSA (500 L, 5 mmol, 2 eq.) and the mixture was refluxed for 24h. The crude was allowed to cool to room temperature and was diluted with aq. NH4Cl saturated solution. The aqueous layer was extracted with EtOAc several times. The organics were dried over Na2SO4, filtered and the solvents were removed. The crude mixture was purified by flash column chromatography (silica, DCM/EtOH 0 to 1.2%) to afford 512 mg of the expected compound 1 (yield 97%). [START_REF] Amabilino | Supramolecular materials[END_REF] A dried Schlenk tube was charged with compound 3 (0.164 g, 0.52 mmol, 1 eq.), compound C (0.870 g, 1.3 mmol, 2.5 eq.), EDC.HCl (0.248 g, 1.3 mmol, 2.5 eq.) and DMAP (0.157 g, 1.3 mmol, 2.5 eq.) under argon flow. Dry DCM (3.5 mL) was added via syringe. The brownish solution was then warmed at 30°C over 3 days. The crude was diluted with DCM, washed with water. The organic phase was collected, dried on Na2SO4, filtered. The solvent was removed. Purification by column chromatography (silica, DCM/EtOAc 100%/0% to 98%/2%) afforded the desired compound (m = 0.671 g, yield 80%).

Compound L 2 .

A round bottom flask equipped with a magnetic stirring bar was charged with compound 4 (0.671 g, 0.41 mmol, 1 eq.). The compound was dissolved in a 1/1 (v/v) of DCM and MeOH (total volume of 30 mL) at room temperature. Then, K2CO3 (2.552 g, 18.5 mmol, 45 eq.) was added and the suspension was stirred 4-5 hrs. The mixture was partitioned between water and DCM. The aqueous layer was extracted several times with DCM. The organic phase was dried on MgSO4, filtered and the solvents were removed to afford a light yellowish powder with enough purity to be used for the next step (0.635 g, yield 99%).

Scheme 1 .

 1 Scheme 1. Synthetic routes to compounds L 1 , L 2 , Ru1 and Ru2.

Figure 1 .

 1 Figure 1. Pictures of gels from a) Ru2 and b) L 2 derivatives. Gel to solution transition temperatures are indicated at the bottom of the figure.

34°C

Figure 2 .

 2 Figure 2. SEM images of xerogels of L 2 in cyclohexane, CGC = 1.3 mg/mL a) scale bar at 5µm, b) scale bar at 1 µm. SEM images of xerogels of Ru2 in benzene, CGC = 30 mg/mL c) scale bar at 5µm, d) scale bar at 1µm.

Figure 3 .

 3 Figure 3. Xerogel FTIR spectrum of L 2 in cyclohexane, CGC = 1.3 mg/mL and of Ru2 in benzene, CGC = 30 mg/mL.

Scheme 3 .Figure 4 .

 34 Scheme 3. Partial 1 H VT-NMR monitoring of Ru2 in C6D6 (c = 30 mg / mL). Inset shows 31 P{ 1 H}VT-NMR monitoring recorded in the same conditions. Lines are used as guide for the eye. Red line = amide protons, green line = methyl signal and purple line = 1,2-dppe signals

Compound 3 .

 3 A dried Schlenk tube was charged with compound A (0.200 g, 0.8 mmol, 1 eq.), compound B (0.208 g, 1.0 mmol, 1.3 eq.), PdCl2(PPh3)2 (0.028 g, 5 mol%) and CuI (0.007 g, 5 mol%) under argon flow. Dry THF (4 mL) and dry DIPA (4 mL) were added by syringe. The mixture darkened. The dark solution was refluxed (65°C) 24 hrs. The crude was diluted with saturated aq. NH4Cl solution and extracted with Et2O several times. The organics were combined, dried over MgSO4, filtered and the solvents were removed. Purification by column chromatography (silica, DCM/EtOAc mixture) afforded the desired compound 3 (m = 0.230 g, yield 90%).[START_REF] Amabilino | Supramolecular materials[END_REF] H NMR (400 MHz, CDCl3): δ = 7.42 (s, 4H, H10/11), 6.38 (s, 2H, H7), 3.59 (broad singlet, 4H, Hamine), 1.97 (s, 3H, H1), 0.26 (s, 9H, HTMS); 13 C NMR (100 MHz, CDCl3): δ = 145.09 (C12), 131.78 (C11b), 131.28 (C11a), 123.69 (C10), 122.47 (C9), 120.77 (C8), 109.73 (C7), 108.35 (C6), 104.73 (C5), 95.98 (C4), 91.99 (C3), 87.35 (C2), 10.33 (C1), -0.09 (CTMS). HRMS (ESI): m/z 341.1445 calcd for C20H22N2Si Na [M+Na + ]; found: 341.1445.Compound 4.

  1 H NMR (400 MHz, CDCl3): δ=7.74 (s, 2H16), 7.65 (br. s, 2H, Hamine), 7.42 (s, 4H, H18), 7.09 (s, 4H,H14), 4.04 (m, 12H, H8), 2.25 (s, 3H, H1), 1.83 (m, 12H, H2-7), 1.43-1.23 (m, 108H, H2-7), 0.88 (m, 18H, H2-7), 0.25 (s, 9H, HTMS); 13 C NMR (75 MHz, CDCl3): δ= 165.94 (C23), 153.28 (C22), 141.77 (C21), 139.67 (C20), 136.43 (C19), 131.83 (C18b), 131.32 (C18a), 128.99 (C17), 125.42 (C16), 123.05 (C15), 123.00 (C14), 106.02 (C13), 104.61 (C12), 96.25 (C11), 90.45 (C10), 89.45 (C8/9), 73.59 (C8/9), 69.44 (C7), 31.92 (C6), 30.36 (C5), 29.75 (C5), 29.71 (C5), 29.66 (C5), 29.61 (C5), 29.44 (C5), 29.36 (C5), 26.11 (C4), 22.68 (C3), 14.09 (C2), 13.36 (C1), -0.10 (CTMS). HRMS (MALDI-DCTB): m/z 1654.29317 calcd for C106H174N2O8SiNa [M+Na + ]; found: 1654.3060. IR (cm -1 ): 3177 (N-H), 2917 (C-H), 2850 (C-H), 2109 (CC), 1635 (C=O), 1580 (C=C).

  (s, 3H, H1), 1.80 (m, 12H, H6), 1.47-1.27 (m, 108H, H4/5), 0.88 (m, 18H, H2); 13 C NMR (75 MHz, CDCl3): δ = 165.96 (C25), 153.26 (C24), 141.70 (C23), 136.40 (C22), 131.99 (C21b), 131.39 (C21a), 128.94 (C20), 127.69 (C19), 125.51 (C18), 123.45 (C17), 121.93 (C16), 121.08 (C15), 105.96 (C14), 90.51 (C13), 89.23 (C12), 83.20 (C11), 78.88 (C10), 73.57 (C9), 69.40 (C8), 31.92 (C7), 30.36 (C6), 29.76 (C5), 29.66 (C5), 29.45 (C5), 29.37 (C5), 29.36 (C5), 26.12 (C4), 22.68 (C3), 14.10 (C2), 13.38 (C1). HRMS (ESI): m/z 1582.2536 calcd for C103H166N2O8Na [M+Na + ]; found: 1582.2543. IR (cm -1 ): 3442 (N-H), 3288CC-H), (2916 (C-H), 2852 (C-H), 2107 (CC), 1635 (C=O), 1583 (C=C). Compound 5.A round bottom flask equipped with a magnetic stirring bar was charged with compound L 2 (0.448 g, 1 mmol, 1 eq.), anhydrous CuCl2 (0.004 g, 0.0287 mmol 0.1 eq.) and THF (6 mL). Neat DBU was added dropwise (0.051 mL, 0.34 mmol, 1.2 eq.). The mixture was refluxed over 1 day with no precaution to remove air. The reaction mixture was taken to dryness. Purification by column chromatography (Florosil®, PE/CHCl3 (9/1, v/v) to CHCl3) followed by precipitation in a mixture of CHCl3 and MeOH afforded the pure compound 5 (m = 0.150 g, yield 17%).[START_REF] Amabilino | Supramolecular materials[END_REF] H NMR (400 MHz, CDCl3): δ = 7.90 (s, 4H, Hamide), 7.64 (s, 4H, HAr tol), (d, 4H, 3 JHH=8.3 Hz, HAr acetylene), 7.37 (d, 4H, 3 JHH =8.3 Hz, HAr acetylene), 7.12 (s, 8H, HAr side), 4.02 (m, 24H, HOmethylene), 2.20 (s, 6H, HMethyl), 1.81 (m, 24H, Hchain), 1.47-1.26 (m, 216H, Hchain), 0.88 (m, 36H, Hchain); 13 C NMR (75 MHz, CDCl3): δ= 166.07, 153.

  [START_REF] Amabilino | Supramolecular materials[END_REF] ): 3181 (N-H), 2916 (C-H), 2054 (C-H), 1636 (C=O), 1583 (C=C). Compound Ru2. A dried Schlenk flask was charged with cis-RuCl2(dppe)2 (0.121 g, 0.13 mmol, 1 eq.), compound L 2 (0.400 g, 0.26 mmol, 2.05 eq.) and NaPF6 (0.122 g, 0.73 mmol, 5.8 eq.) under argon flow. The tube was evacuated and backfilled with argon. Then, a solution of distillated Et3N in dry DCM (16 mL) was added. Finally, the reaction mixture was warmed at 30°C during 4 days.[START_REF] Wu | Availability, Applicability, and Advanceability[END_REF] P{ 1 H} (no lock procedure) NMR monitoring indicated only one singlet signal at 52.8 ppm. The reaction was stopped by adding 20 mL of MeOH over 20 min. The compound was filtered-off. The powder was taken up in DCM (20 mL) and precipitate with MeOH (8 mL) to afford compound Ru2 pure as a yellow powder (m = 0.412 g, yield 82%). 1 H NMR (400 MHz, CD2Cl2): δ =7.74 (s, 4H, Hamide), 7.73 (s, 4H, Htol), 7.49 (m, 16H, Hdppe), 7.30 (d, 4H, 3 JHH = 8.3 Hz, HAr acetylene), 7.20 (s, 8H, 3 JHH = 7.5 Hz, Hdppe), 6.97 (t, 16H, 3 JHH = 7.7 Hz, Hdppe), 6.72 (d, 4H, 3 JHH = 8.4 Hz, HAr acetylene), 4.04 (dt, 24H, 3 JHH = 6.6 Hz, HOmethylene), 2.66 (br. s, 8H, Hdppe), 2.25 (s, 6H, HMe), 1.88-1.71 (m, 28H, Haliphatic), 1.54 (m, Haliphatic), 1.28 (br. s, 205H, Haliphatic), 0.88 (m, 36H, Haliphatic); 13 C NMR (75 MHz, CD2Cl2): δ = 165.91, 153.

H

  ), 2921 (C-H), 2054 (CC), 1636 (C=O), 1583 (C=C). 𝜆max (): 402 nm (solvent = DCM, 63 000 L mol -1 cm -1 ).

Table 1 . Summary of gelation properties

 1

		Cy	MeCy	n-heptane	n-dodecane	benzene	toluene	o-xylene	m-xylene
	L2	G (1.3)	G (4.0)	G (1.3)	G (1.4)	NS	NS	NS	NS
	5	NS	NS	NS	NS	NS	NS	NS	NS
	Ru1	P	P	NS	NS	S	S	S	S
	Ru2	NS	NS	NS	NS	G (30)	G (

  br. m.),26.59, 23.14, 23.12, 14.32, 13.13. 31 P{ 1 H} NMR (162 MHz, CD2Cl2): δ = 53.17. HRMS (ESI): m/z 3814.62505 calcd for C242H370N4O16P4 102 Ru [M +. ]; found 3814.6298. IR (cm -1 ): 3255 (N-H), 2912, 2851 (C-H), 2051 (CC), 1639 (C=O), 1582 (C=C). 𝜆max (solvent, ): 338 nm (DCM, 38 000 L mol -1 cm -1 ).

	H
	NMR (400 MHz, CDCl3): δ = 6.33 (s, 2HAr), 3.53 (broad singlet,
	4Hamine), 1.95 (s, 3Hmethyl), 0.22 (s, 9HTMS); 13 C NMR (100 MHz,
	CDCl3): δ = 144.84, 120.92, 110.25, 108.47, 105.74, 92.08, 10.31, 0.02
	(CTMS). HRMS (ESI): m/z 219.1312 calcd for C12H19N2Si [M+H + ];
	found: 219.1312.
	Compound Ru1. A dried Schlenk flask was charged with cis-
	RuCl2(dppe)2 (0.050 g, 0.05 mmol, 1 eq.), compound L 1 (0.165 g, 0.11
	mmol, 2.2 eq.) and NaPF6 (0.052 g, 0.31 mmol, 6 eq.) under argon flow.
	The tube was evacuated (30 min) and backfilled with argon. Then, a
	solution of distillated Et3N (417 L, 3 mmol, 60 eq.) in dry DCM (16
	mL) was added with a ssyringe., The reaction mixture was stirred at
	30°C during 4 days. 31 P{ 1 H} (no lock procedure) NMR monitoring of
	the reaction indicated only one singlet signal at around 53 ppm. The
	reaction mixture was diluted with DCM (10 mL), washed with water
	(3 × 10 mL). The organic layer was collected dried on Na2SO4, filtered
	and the solvent was removed. The crude was taken up in 10 mL DCM
	and precipitated by dropwise addition of 10 mL of MeOH. The yellow
	precipitate was filtered-off under air and dried to afford the
	corresponding Ru1 compound (m=0.164 g, yield 83%). 1 H NMR (400
	MHz, CD2Cl2): δ =7.51 (m, 20H Hdppe + Hamide), 7.49 (t, 16H, 3 JHH =
	7.6 Hz, Hdppe), 7.13-7.17 (m, 16H, HAr+Hdppe), 6.81 (s, 4H, Htol), 4.08
	(dt, 24H, 3 JHH=6.5 Hz, Halkyl), 2.66 (br. s, 8H, CH2dppe), 2.17 (s, 6H,
	HMe), 1.88-1.71 (m, 28H, Hchain), 1.54 (m, Hchain), 1.28 (br. s, 205H,
	Hchain), 0.88 (m, 36H, MeChain); 13 C NMR (75 MHz, CD2Cl2): δ =
	166.09, 153.75, 141.68, 141.68, 137.46 (quint, J=10.2 Hz), 136.27,
	134.61, 130.43, 129.09, 127.57, 125.24, 123.13, 116.07, 73.93, 69.82,
	32.39, 31.85 (m, 1 JP,C + 3 JP,C = 24 Hz, P(CH2)2P), 30.81, 30.21-29.81

(

  24, 141.73, 136.40, 132.32, 131.48, 128.91, 128.13, 125.83, 123.90, 121.45, 121.05, 106.06, 91.28, 89.35, 82.01, 75.72, 73.56, 69.41, 31.82, 30.38, 29.76, 29.67, 29.47, 29.37, 26.13, 22.68, 14.08, 13.36. HRMS (ESI): m/z 3139.5024 calcd for C206H330N4O16Na [M+Na + ]; found: 3139.5007. IR (cm -

  27, 141.47, 137.17, 137.00, 136.87, 136.69, 134.10, 130.89, 130.76, 129.99, 129.83, 129.23, 128.74, 127.77, 125.29, 121.72, 117.63, 116.58, 105.82, 90.93, 88.53, 73.49, 69.37, 31.94, 31.38 (m, 1 JP,C + 3 JP,C = 24 Hz, P(CH2)2P), 30.40, 29.77-29.38 (br. m.), 26.16, 22.69, 13.87, 13.24. 31 P{ 1 H} NMR (162 MHz, CD2Cl2): δ = 52.89. HRMS (ESI): m/z 1338.2289 calcd for C258H378N4O16P4 102 Ru [M 3+. ]; found: 1338.2321. IR (cm -1 ): 3432 (N-

ACKNOWLEDGMENT

The authors thank the Université de Rennes 1 and the CNRS for support. The authors are grateful to Dr. Camerel for the fruitful discussions about the gel formation. This work was granted access to the computing resources of CINES (Montpellier, allocation 2019-A0060810728 and 2020-AP010811752 awarded by GENCI). The authors also thank F. Gouttefangeas and L. Joanny from the CMEBA (Centre de Microscopie Electronique à Balayage et microAnalyse, Rennes) for assistance in recording SEM images.

ASSOCIATED CONTENT Supporting Information

The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION

Corresponding Author * olivier.galangau@univ-rennes1.fr * stephane.rigaut@univ-rennes1.fr

Notes

The authors declare no competing financial interest.


Thomas Castaing-Cordier

Virginie Ladroue

Fabrice Besacier

Audrey Bulete

Denis Jacquemin

Patrick Giraudeau

Jonathan Farjon
email: jonathan.farjon@univ-nantes.fr

High-field and benchtop NMR spectroscopy for the characterization of new psychoactive substances

Keywords: Elucidation Quantification Identification New Psychoactive Substances, High Field NMR spectroscopy, benchtop NMR spectrometer, structure elucidation, quantification

New psychoactive substances (NPS) have become a serious threat for public health in Europe due to their ability to be sold in the street or on the darknet. Regulating NPS is an urgent priority but comes with a number of analytical challenges since they are structurally close to legal products. A number of analytical techniques can be used for identifying NPS, among which NMR spectroscopy is in the limelight. High field NMR is typically used for structural elucidation in combination with others techniques like GC-MS, Infrared spectroscopy, together with databases. In addition to their strong ability to elucidate molecular structures, high field NMR techniques are the gold standard for quantification without any physical isolation procedure and with a single internal standard. However, high field NMR remains expensive and emerging "benchtop" NMR apparatus which are cheaper and transportable can be considered as valuable alternatives to high field NMR. Indeed, benchtop NMR, which has emerged about ten years ago, makes it possible to carry out structural elucidation and quantification of new psychoactive substances despite the gap in resolution and sensitivity as compared to high field NMR. This review describes recent advances in the field of NMR applied to the characterization of NPS. High-field NMR methods are first described in view of their complementarity with other analytical methods, focusing on both structural and quantitative aspects. The second part of the review highlights how emerging benchtop NMR approaches could act as a game changer in the field of forensics.

 New psychoactive substances are a serious threat for public health  These drugs are sold in the street or the darknet and need an urgent identification  Nuclear Magnetic Resonance is a gold standard for elucidation and quantification  Complementarity of other analytical techniques is essential to elucidate drugs  Benchtop Nuclear Magnetic Resonance is an emerging mobile approach for forensics

Introduction

New psychoactive substances (NPS) gather different families of molecules such as aminoindanes, synthetic cannabinoids, synthetic cathinones, phenethylamnines, piperazines, tryptamines, and plant-based substances (Figure 1) [START_REF]What are NPS?[END_REF]. NPS are molecules derived from scheduled or commercial drugs which have been misused or whose structure has been slightly modified. As a consequence of these chemical modifications, these hazardous compounds are not always controlled under the International Drug Control Conventions, and their legal status remains undefined. Since 1997, 450 NPS have been reported in Europe, and among them two thirds since 2008 [START_REF]Nouveaux produits de synthèse -Synthèse des connaissances -OFDT[END_REF]. NPS are usually purchased on the darknet but also in the street [START_REF] Marino | Rapid Identification of Synthetic Cannabinoids in Herbal Incenses with DART-MS and NMR[END_REF]. The growing occurrence of these molecules on the European market is an alarming threat for public health because they are widely consumed during festive events as recreational drugs and their use could lead to death by overdose. In this context, there is an urgent need to develop robust and high-throughput analytical tools to help reporting NPS. In particular, methods are needed to identify the molecular structure of known NPS and to elucidate the structure of unknown compounds that appear on the market, either as pure molecules or in mixtures.

Figure 1: Various Spice products (a) "Mountain Industry"; (b) JWH-122 powder; "Moon Spice", leaf; (c) "Barely Legal", "Spice" package; (d) "Melon Code Black", "Spice" package. Reproduced from Ref. [START_REF] Marino | Rapid Identification of Synthetic Cannabinoids in Herbal Incenses with DART-MS and NMR[END_REF] with permission.

Several experimental techniques are commonly used to characterize the structure of NPS. First, InfraRed (IR) and Raman spectroscopies, the most widespread optical techniques, are tools of choice to determine the chemical functions of the molecule under study [START_REF] Omar | Identification of new psychoactive substances (NPS) by Raman spectroscopy[END_REF]. In addition, different variants of Mass Spectrometry (MS) coupled to chromatography are also widely used. MS, with Chemical Ionization (CI) is used to provide the molecular weight [START_REF] Elie | Keeping pace with NPS releases: fast GC-MS screening of legal high products[END_REF]. In addition, MS with Electronic Impact (EI) mode is able to provide information on different fragments of the molecule [START_REF] Elie | Keeping pace with NPS releases: fast GC-MS screening of legal high products[END_REF]. Hyphenated MS-MS or MS² makes it possible to get further insight into fragmentation schemes of the molecule [START_REF] Vicente | Systematic analytical characterization of new psychoactive substances: A case study[END_REF]. Finally, High Resolution Mass Spectrometry (HRMS) allows for getting a high resolved molecular weight of the compound [START_REF] Rossi | An analytical approach to the forensic identification of different classes of new psychoactive substances (NPSs) in seized materials[END_REF]. In order to achieve a faster structural elucidation, most of the above-mentioned techniques are combined with databases [START_REF] Dçebska | Spectral Databases, Infrared[END_REF][START_REF] Ausloos | The critical evaluation of a comprehensive mass spectral library[END_REF]. In addition, structural elucidation is sometimes supported by Density Functional Theory (DFT) calculations to predict spectroscopic values, e.g., vibrational frequencies in IR [START_REF] Ali | DFT Study for the Spectroscopic and Structural Analysis of p-Dimethylaminoazobenzene[END_REF] or NMR chemical shifts and couplings [START_REF] Bagno | Predicting 13C NMR Spectra by DFT Calculations[END_REF] in order to help confirming the final structure. Finally, one of the most suitable techniques for a complete molecular structure elucidation is Nuclear Magnetic Resonance (NMR) spectroscopy. Indeed, NMR spectroscopy offers accurate information on the chemical environment of all atoms in a molecule through chemical shifts, while providing crucial input on atomic connectivities through J-couplings.

There are two possible scenarios in which NMR can contribute. If the NPS is already known, the guidelines from the scientific working group for the analysis of the seized drugs (SWGDRUGS) present a resourceful strategy to reach identification [START_REF]SWGDRUG Approved Recommendations[END_REF]. They categorize analytical techniques into three groups depending on the level of selectivity they are able to achieve in the identification and quantification of pure or mixed analytes. Category A techniques provide the highest level of selectivity through structural information. Category B techniques provide an intermediate level of selectivity through physical or chemical characteristics without structural information whereas category C techniques give rise to a low level of selectivity but provide general or chemical class information. Unambiguous structure identification requires combining two categories: at least one category A technique with another from category A, B, or C. All analytical results need to be compared with those that are obtained from reference materials. For example, the analytical workflow may contain GC and MS from category B and A respectively, or NMR with melting point which are from category A and C, respectively. If the substance is totally unknown, it could be necessary to carry out a complete structural elucidation to confirm or disprove the illicit character of the molecule being studied. NMR can be a technique of choice in such case thanks to its strong structure elucidation capabilities. However, NMR is not commonly used by customs, police, or forensic labs because it is expensive to purchase and maintain, notably due to the use of cryogenic fluids. Moreover, the instrument is bulky and requires dedicated staff for maintenance.

In addition to the role NMR can play in the identification of NPS, it also has the asset of allowing quantification without resorting to a reference material identical or structurally close to the analyte. In the case of emerging molecules, such reference materials happen to be either expensive or simply not available yet.

In this context, the present review aims at highlighting the role of NMR as an analytical tool for the study of NPS, focusing both on high-field NMR and on emerging benchtop devices. Both identification and quantitation capabilities of NMR are described, as well as its complementarity to other analytical approaches in NPS structure elucidation workflows. NMR has great ability to identify and quantify small organic molecules, and is therefore essential for the structure elucidation of NPS. A first strategy consists in associating NMR spectra with databases [START_REF] Ali | DFT Study for the Spectroscopic and Structural Analysis of p-Dimethylaminoazobenzene[END_REF]. This is particularly useful when a known molecule is suspected, but also to identify compounds in mixtures whose spectra are overlapped. In the case of unknown NPS, as for many other molecules, identification of the structure by NMR relies on a set of complementary experiments rather than on NMR alone [START_REF] Shevyrin | Synthetic cannabinoid 3-benzyl-5-[1-(2pyrrolidin-1-ylethyl)-1H-indol-3-yl]-1,2,4-oxadiazole. The first detection in illicit market of new psychoactive substances[END_REF]. Figure 2 shows the typical flowchart used for the structural elucidation of NPS, it is used here to support the methodology. Note that it also includes quantification aspects that are described in the next section. Structural analysis typically starts with the identification of major fragments, the determination of the molecular weight and/or the molecular formula by MS, MS² or by GC/LC-MS [START_REF] Vicente | Systematic analytical characterization of new psychoactive substances: A case study[END_REF]. It can be complemented by the acquisition of IR or Raman spectra to determine the main chemical functions present in the molecule [START_REF] Angerer | Separation and structural characterization of the new synthetic cannabinoid JWH-018 cyclohexyl methyl derivative NE-CHMIMO using flash chromatography, GC-MS, IR and NMR spectroscopy[END_REF]. In parallel, NMR analysis makes it possible to determine the exact structure of the molecule or to confirm results obtained by chromatography and MS². One dimensional (1D) proton-1 ( 1 H) spectra are often supplemented by carbon-13 ( 13 C) spectra through direct or indirect detection. The latter, based on DEPT or INEPT experiments, offers higher sensitivity and spectral editing information allowing to identify the degree of protonation of carbons, while the former, although less sensitive, is required to detect quaternary carbons. In addition, in the case of mixtures, the larger spectral range of 13 C can help improve separating molecules whose 1 H NMR signals are overlapped. Then, the acquisition of 2D spectra provides information on through-bond or space atomic connectivities. Most popular experiments include 1 H-1 H homonuclear COrrelated SpectroscopY (COSY) to determine the neighboring 1 H. Next, 1 H- 13 C Heteronuclear bidimensional (2D) experiments such as Heteronuclear Single Quantum Correlation (HSQC) help determining the 13 C directly bearing 1 H in the molecule. Finally, 2D 1 H- 13 C Heteronuclear Multiple Bond Correlation (HMBC) experiments correlate long range 1 H and 13 C that are separated by two, three and four bonds (Figure 2). Despite the lower sensitivity of heteronuclear experiments coming from the selection of 1 H- 13 C isotopomers at natural abundance, these techniques are the most informative for the structure since they correlate short and long range coupled 1 H and 13 C nuclei in the molecule. When the molecule is flexible, it may be important to access its 3D structure, which is possible through the Nuclear Overhauser Effect SpectroscopY (NOESY) or the Rotating frame Overhauser Effect SpectroscopY (ROESY) techniques [START_REF] Angerer | Separation and structural characterization of the new synthetic cannabinoid JWH-018 cyclohexyl methyl derivative NE-CHMIMO using flash chromatography, GC-MS, IR and NMR spectroscopy[END_REF]. For instance, those techniques can be used to determine the Z or E configuration of an alkene, the relative configuration of asymmetric carbons (R, S), while they can also provide evidence for the conformational mobility of the structure. The above mentioned workflows have been applied multiple times to elucidate the structure of NPS. While chromatography and mass spectrometry are most often used in complementarity with NMR, IR and Raman spectroscopies are rather used as optional tools, and little details are given in the literature [START_REF] Angerer | Separation and structural characterization of the new synthetic cannabinoid JWH-018 cyclohexyl methyl derivative NE-CHMIMO using flash chromatography, GC-MS, IR and NMR spectroscopy[END_REF][START_REF] Westphal | Mass, NMR and IR spectroscopic characterization of pentedrone and pentylone and identification of their isocathinone by-products[END_REF][START_REF] Böselt | Determining the Regiochemistry and Relative Stereochemistry of Small and Druglike Molecules Using an Alignment Algorithm for Infrared Spectra[END_REF].

High Field NMR 2.1 NMR for structural elucidation

1D 1 H, 13 First, NMR can be used to identify a molecule or a mixture of molecules. This approach was reported by Gibbons et al. [START_REF] Gibbons | An analysis of the 'legal high' mephedrone[END_REF] to identify compounds in a legal high, i.e., a NPS from plant or fungal material. ESI HRMS was first used to provide the accurate molecular weight hence the exact molecular formula. Then, 1 H and 13 C NMR spectra were applied to determine the purity of NPS. Finally, 2D pulse sequences were acquired for an unambiguous assignment of all carbons and hydrogens of the molecule leading to mephedrone. In another study, Lobo Vicente et al. implemented the entire workflow sketched in Figure 2. They took advantage of GC-MS, FTIR, NMR, HR-MS/MS and Raman spectroscopy on two powders found in a box from China, and the NPS could be identified as 5F-AMB and PX-3, two synthetic cannabinoids [START_REF] Vicente | Systematic analytical characterization of new psychoactive substances: A case study[END_REF].

In addition to this typical workflow, NMR is often used to differentiate isomers of constitution, i.e., molecules having the same molecular formula but different structures. This approach is particularly useful to elucidate NPS in seized samples since traffickers are diverting known active moieties into unscheduled molecules, by creating small cheap structural modifications like isomerizations. For instance, Marino et al. used 1 H NMR in combination with Direct Analysis in Real Time Mass Spectrometry (DART-MS) to determine the structure of synthetic cannabinoids in herbals [START_REF] Marino | Rapid Identification of Synthetic Cannabinoids in Herbal Incenses with DART-MS and NMR[END_REF]. DART-MS produced a spectrum of the molecular ions indicating how many species were present in the sample, and their fragmentation schemes allowed confirming the presence of specific features as well as their molecular formula thanks to the exact mass. DART-MS allows to work directly on the sample with no need to dissolve it, however this technique is not always able to differentiate two isomers with identical fragments. Thus, in this example, the authors were able to reveal a naphtyl atropisomerism by comparing the NMR spectral fingerprints of herbal products containing NPS powder and pure standards. Strano et al. [START_REF] Rossi | An analytical approach to the forensic identification of different classes of new psychoactive substances (NPSs) in seized materials[END_REF] applied a similar strategy to identify different classes of seized drugs. In this case, the differentiation of positional isomers of aminopropyl was carried out with the help of JHH coupling constants in the aromatic 1 H spectral region.

In some cases, the information obtained from 1 H NMR spectra can be limited by spectral overlap resulting from complex mixtures. Nevertheless, Bottineli et al. [START_REF] Bottinelli | GC-QTOF and NMR analyses to elucidate composition of 41 powders from an NPS collector[END_REF] showed that 1D 1 H and 13 C acquisitions made it possible to identify 4-FA and 2-FMA which are one of the three positional isomers of fluoroamphetamine and fluoromethamphetamine, respectively. In most cases, it is preferable to rely on 2D correlation-based experiments. For instance, Westphal et al. showed how COSY made it possible to locate the position of the methylenedioxy group of pentylone but also the unbranched side chain of pentedrone [START_REF] Westphal | Mass, NMR and IR spectroscopic characterization of pentedrone and pentylone and identification of their isocathinone by-products[END_REF]. The same authors also demonstrated that the COSY map allows the identification of the position of the alkyl chain for isopentedrone. While 2D homonuclear experiments facilitate structural elucidation, there are even more resolutive and informative techniques, such as 13 C based-heteronuclear acquisitions. For example, Angerer et al. made a full characterization of a new synthetic cannabinoid called JWH-018 by recording a series of NMR spectra: 1D-1 H, 13 C NMR, 1D-Total Correlated SpectroscopY (TOCSY), 1D-ROESY, 2D 1 H-1 H COSY, 1 H-13 C HSQC, and 1 H- 13 C HMBC [START_REF] Angerer | Separation and structural characterization of the new synthetic cannabinoid JWH-018 cyclohexyl methyl derivative NE-CHMIMO using flash chromatography, GC-MS, IR and NMR spectroscopy[END_REF]. In the case of NPS, 2D NOESY was also used to determine the positional isomer (out of six possibilities) of a previously unknown molecule [START_REF] Westphal | Structure elucidation of a new open chain isomer of the cannabimimetic cyclopropoylindole A-796[END_REF] or to prove a iodo substitution [START_REF] Jankovics | Detection and identification of the new potential synthetic cannabinoids 1-pentyl-3-(2-iodobenzoyl)indole and 1-pentyl-3-(1-adamantoyl)indole in seized bulk powders in Hungary[END_REF].

NMR as an accurate quantitative tool

In parallel to structural elucidation, NMR has the ability to accurately quantify the different compounds present in NPS samples. Quantification of NPS can bring important information on sample origin, especially when the active molecule is mixed with other NPS or cutting agents such as ibuprofen, caffeine, sugars, etc. [START_REF] Hays | Proton Nuclear Magnetic Resonance Spectroscopy (NMR) Methods for Determining the Purity of Reference Drug Standards and Illicit Forensic Drug Seizures[END_REF]. In addition, it allows official organizations such as EMCDDA (European Monitoring Centre for Drugs and Drug Addiction) to warn about the toxicity of the products and to track the drug trafficking networks. Chromatography coupled with MS is the gold standard analytical method in forensic laboratories for the quantification of seized samples of illicit drugs. However, NMR stands as an interesting alternative since: i) quantification can be achieved without physical separation of the sample components; ii) multiple compounds can be quantified with a single internal reference which does not necessarily have to be close to the analyte in contrast to chromatography; and iii) NMR yields a better accuracy, typically 2% vs 6% for GC [START_REF] Almeida | A Validated NMR Approach for MDMA Quantification in Ecstasy Tablets[END_REF] (cf. Figure 2). There are several possibilities to quantify molecules in a sample. The mass or concentration of a compound can be determined from the reference concentration:

Amount (mg g) ⁄ = [REF] × 𝐴 x 𝐴 REF × 𝑁 REF 𝑁 x × 𝑉 × 𝑀 x 𝑚 sample (eq. 1)
in which, [REF] is the concentration of the internal standard (mol/L), Ax the integral of the analyte signal, AREF the integral of the reference signal, NREF the number of reference protons, Nx the number of analyte protons, V the volume of the solution (L), Mx the molecular weight of the analyte (mg/mol), and msample the mass of sample (g).

Moreover, it is also possible to determine the purity by taking into account the purity of the reference:

𝑃 sample = 𝐴 x 𝐴 REF × 𝑁 REF 𝑁 x × 𝑀 x 𝑀 REF × 𝑚 REF 𝑚 sample × 𝑃 REF , (eq. 2)
With Psample the purity of the sample as mass fraction (%) and PREF the purity of the reference as mass fraction (%).

These strategies have been used in different studies involving NPS. For instance, Eq. 1 was applied to precisely determine amounts of synthetic cannabinoids in a complex herbal mixtures [START_REF] Assemat | Screening of "spice" herbal mixtures: From high-field to low-field proton NMR[END_REF]. Concentrations were determined by comparing the areas of targeted protons to the ones of dimethyl maleate as an internal reference with precise known concentration. NMR was also applied to determine the mass of psychoactive product in a tablet. Naqi et al. quantified seized MDMA by using 1 H-qNMR, UHPLC and UHPLC-MS (Table 1) [START_REF] Naqi | 1H quantitative NMR and UHPLC-MS analysis of seized MDMA/NPS mixtures and tablets from night-club venues[END_REF]. Maleic acid and MDMA-d5 were used respectively as an internal reference for NMR and UHPLC measurements. The MDMA concentrations determined by UHPLC and NMR were found comparable with no significant statistical difference as revealed by ANOVA single factor analysis. In another study, Eq. 2 allowed determining the purity of the compounds present in mixtures of NPS or cutting agents (vitamins, caffeine, etc.) [START_REF] Gaspar | 4F-PBP (4′-fluoro-α-pyrrolidinobutyrophenone), a new substance of abuse: Structural characterization and purity NMR profiling[END_REF]. Finally, the case of a 41-yearold man known as a polydrug addict and found dead at home was resolved by resorting to NMR [START_REF] Ameline | Identification and analytical characterization of seven NPS, by combination of 1H NMR spectroscopy, GC-MS and UPLC-MS/MS®, to resolve a complex toxicological fatal case[END_REF]. Seven small bags of unknown powders and crystals, suspected to be NPS, were found close to the body and sent to the laboratory 3 weeks later. A complete structural elucidation was carried out with 1D 1 H and 13 C spectra, DEPT 13 C spectra, and a set of 2D experiments including 1 H-1 H COSY, edited 1 H-13 C HSQC, and 1 H- 13 C HMBC. 19 F experiments were performed as well. Moreover, NPS were quantified and their purity was determined thanks to Eq. 2 using maleic acid as an internal reference. This investigation revealed that the purity of the recovered compounds was between 50.8 and 89.5%, which is within the usual range of products seized in Europe [START_REF]Observatoire Français des Drogues et des Toxicomanies, Drogues, chiffres clés[END_REF][START_REF]European Monitoring Center for Drugs and Drug Addiction, European Drug Report: Trends and Developments[END_REF].

In short, NMR can be seen as a new gold standard for quantification since it does not require the use of a structurally close reference to the analyte and leads to a lower RSD than chromatography. In addition, it remains a non-destructive technique and allows to quantify multiple compounds in a sample at the same time.

Benchtop NMR spectroscopy 3.1. Advantages and drawbacks

Despite the above-mentioned advantages, NMR is not yet a routine tool in forensics due to both the bulky and expensive high-field equipment and to the need to fill magnets with cryogenic fluids. An alternative could emerge from medium-field benchtop NMR spectrometers that do not suffer from such drawbacks. Benchtop NMR spectroscopy represents a major advance in quality control since it is transportable and can therefore be directly positioned in production sites. It has already been used for the study of adulteration of oils [START_REF] Parker | 60MHz 1H NMR spectroscopy for the analysis of edible oils[END_REF], dietary supplements [START_REF] Pagès | Evaluation of a Benchtop Cryogen-Free Low-Field 1H NMR Spectrometer for the Analysis of Sexual Enhancement and Weight Loss Dietary Supplements Adulterated with Pharmaceutical Substances[END_REF] and meat [START_REF] Jakes | Authentication of beef versus horse meat using 60MHz 1H NMR spectroscopy[END_REF], but also to monitor chemical reactions [START_REF] Gouilleux | Real-time reaction monitoring by ultrafast 2D NMR on a benchtop spectrometer[END_REF] or bioprocesses [START_REF] Bouillaud | Benchtop flow NMR spectroscopy as an online device for the in vivo monitoring of lipid accumulation in microalgae[END_REF]. Whereas high field NMR instruments operate above 300 MHz, commercial compact NMR spectrometers operate at 1 H frequencies between 40 and 100 MHz. Moreover, these benchtop spectrometers are characterized by excellent homogeneity, comparable to the one of high-field spectrometers. However, two issues stem from such low magnetic fields: a lower sensitivity but also a reduction of the spectral resolution arising from the much lower spectral width in Hz. Figure 4 illustrates how lower magnetic fields lead to poorly resolved spectra, making it difficult to identify multiplet patterns. This issue is further complicated by second order coupling effects that appear when the chemical shift difference in Hz between coupled spins becomes of the same order of magnitude as the coupling constant between those sites. Second order couplings strongly distort multiplet intensities and increase when the magnetic field decreases, since the resonance frequencies become closer whilst the J-couplings are invariant with magnetic field.

Application to NPS

To the best of our knowledge, no full structural elucidation of NPS has been reported yet on a benchtop spectrometer. However, some results show that for some average-sized compound like strychnine, structure determination is feasible. In reference [START_REF] Singh | Desktop NMR for structure elucidation and identification of strychnine adulteration[END_REF], authors showed that by recording of 1D 1 H, 1D 13 C, 2D J-resolved, 1 H-1 H COSY, 1 H- 13 C HETeronuclear CORrelation (HETCOR), 1 H-13 C HSQC, and 1 H- 13 C HMBC spectra, the redundant information counterbalances the lack of information induced by the low resolution (Figure 5). Molecular databases could offer a complementary efficient strategy to circumvent spectral resolution issues that prevent the use of benchtop NMR to elucidate the structure of NPS. A convincing example relates to the development of an automated algorithm for spectral recognition of NPS [START_REF] Antonides | Rapid Identification of Novel Psychoactive and Other Controlled Substances Using Low-Field 1H NMR Spectroscopy[END_REF]. 1 H NMR spectra of reference compounds were collected using an 80 MHz instrument. The reference library consists of 302 spectra altogether for different classes of NPS. Then 432 seized samples were analyzed by NMR and GC-MS for cross validation. 1 H NMR analysis nicely matched the GC-MS results for 93% of the cases. This example highlights the relevance of combining benchtop NMR with databases, and that such combination can be made even more efficient when databases encompass high field NMR data. Capitalizing on such a strategy, Duffy et al. analyzed several fentanyls at different magnetic fields [START_REF] Duffy | Differentiation of fentanyl analogues by low-field NMR spectroscopy[END_REF]. In order to deal with second order coupling effects that make the comparison of data acquired at different fields challenging, these authors relied on a Quantum Mechanical Spectral Analysis (QMSA) to transform spectra obtained at high field into their benchtop NMR counterpart. This approach was efficient in retrieving structural data from benchtop spectra despite signal overlaps and strong couplings. It also highlights the trends going to the generalization of databases for NPS structure elucidation.

Quantification is also possible with medium-field NMR, although rendered more difficult by strong peak overlaps. Under this situation, it becomes necessary to use an internal reference having isolated characteristic signals. In the field of pharmaceutics, active substances such as acetaminophen, aspirin, diclofenac, ibuprofen, and naproxen were quantified by NMR spectroscopy and compared to those obtained by LC-QTOF-MS (Table 2). The two techniques offered comparable results with a better accuracy for NMR than for LC-QTOF-MS in most cases [START_REF] Keizers | Benchtop NMR spectroscopy in the analysis of substandard and falsified medicines as well as illegal drugs[END_REF].

Table 2: Quantitation of over-the-counter pharmaceutical products, using benchtop 1 H-NMR spectroscopy, using 3,4,5-trichloropyridine as internal standard and LC-QTOF-MS. The NMR analyses were performed with five independent measurements, the LC-MS analyses were performed with two dose units measured in duplicate. Reprinted from [START_REF] Keizers | Benchtop NMR spectroscopy in the analysis of substandard and falsified medicines as well as illegal drugs[END_REF] with permission from Elsevier. This approach could therefore be transposed to the quantification of NPS with no need to find a chromatographic reference from the same family than the molecule of interest.

Perspectives

While benchtop NMR spectroscopy offers promising perspectives for structure elucidation and quantification of NPS at a reasonable cost, the applicability of this technique remains limited by the above-mentioned resolution and sensitivity issues. Promising perspectives could arise from pulse sequence developments that have recently emerged to improve the performance of benchtop devices, based on the addition of magnetic field gradient coil an essential ingredient of pulse sequence developments in high-field NMR for decades. Such developments include ultrafast 2D NMR [START_REF] Gouilleux | High-throughput authentication of edible oils with benchtop Ultrafast 2D NMR[END_REF] allowing the acquisition of singlescan 2D spectra, pure shift sequences [START_REF] Castaing-Cordier | Highly Resolved Pure-Shift Spectra on a Compact NMR Spectrometer[END_REF] enabling to remove JHH couplings, or 2D DOSY [START_REF] Assemat | Diffusionordered spectroscopy on a benchtop spectrometer for drug analysis[END_REF] which provides virtual separation of mixture components based on their diffusion coefficients. Moreover, hyperpolarization techniques parahydrogen based methods [START_REF] Robinson | Hyperpolarised 1H-13C benchtop NMR spectroscopy[END_REF][START_REF] Robertson | Hyperpolarization of Pyridyl Fentalogues by Signal Amplification By Reversible Exchange (SABRE)[END_REF] could also be advantageous in order to reach spectra with an overall better sensitivity for investigating lower amounts of NPS in mixtures, although routine application would require a level of automation that is currently not reach with such methods.

DFT-based simulations [START_REF] Gerrard | IMPRESSION -prediction of NMR parameters for 3-dimensional chemical structures using machine learning with near quantum chemical accuracy[END_REF] may also play a role in facilitating structural elucidation at low magnetic fields. DFT methods are a subgroup of ab initio theories generally considered as the best in terms of accuracy/cost ratio. DFT allows calculating chemical shifts and scalar couplings for all atoms of a given molecule in one shot, possibly accounting for solvent effects during the calculations. DFT can also be combined with machine learning algorithms to reduce the time needed to predict chemical shifts [START_REF] Gerrard | IMPRESSION -prediction of NMR parameters for 3-dimensional chemical structures using machine learning with near quantum chemical accuracy[END_REF].

Conclusions

NMR spectroscopy plays a strategic role in the characterization and quantification of NPS. Most of the time, NMR is used in combination with other analytical techniques to cross-check, confirm, and validate integrated reliable data regarding the molecular structure. Characterization usually requires chromatography coupled with mass spectrometry to determine the molecular weight and the molecular formula of the structure as a starting point to perform any NMR elucidation. Moreover, IR could also bring complementary data of interest to confirm the presence of specific chemical functions.

Benchtop NMR could act as a new game changer in the analysis of NPS in police, customs and forensic laboratories despite its lower resolution. Indeed, it is associated with lower purchase and operating costs, allowing more laboratories to be equipped with NMR. However, benchtop NMR requires pushing further the development of new acquisition methods and integrating NMR with other analytical methods, DFT calculations, and/or databases.

A foreseen analytical workflow (Figure 6) would include the use of a portable platform with medium field NMR equipped with a gradient coil for implementing advanced methods on an 80 to 100 MHz benchtop NMR spectrometer, together with DART-MS, and IR. This integrated platform could be enhanced with databases of all NPS encountered throughout the world as well as machine learning prediction to improve accuracy and speed of elucidation.

Figure 2 :

 2 Figure 2: Flow Chart for structural and quantitative NPS analysis. Straight rectangles describe analytical tools, rounded rectangles describe structural information obtained and rhombuses describe quantitative information.

Figure 3

 3 Figure3details the NMR part of this structural elucidation workflow. Commonly, standard 1D and 2D experiments are needed for the complete structural characterization of the molecule. One dimensional (1D) proton-1 ( 1 H) spectra are often supplemented by carbon-13 ( 13 C) spectra through direct or indirect detection. The latter, based on DEPT or INEPT experiments, offers higher sensitivity and spectral editing information allowing to identify the degree of protonation of carbons, while the former, although less sensitive, is required to detect quaternary carbons. In addition, in the case of mixtures, the larger spectral range of13 C can help improve separating molecules whose 1 H NMR signals are overlapped. Then, the acquisition of 2D spectra provides information on through-bond or space atomic connectivities. Most popular experiments include 1 H-1 H homonuclear COrrelated SpectroscopY (COSY) to determine the neighboring 1 H. Next, 1 H-13 C Heteronuclear bidimensional (2D) experiments such as Heteronuclear Single Quantum Correlation (HSQC) help determining the13 C directly bearing1 H in the molecule. Finally, 2D 1 H-13 C Heteronuclear Multiple Bond Correlation (HMBC) experiments correlate long range 1 H and13 C that are separated by two, three and four bonds (Figure2). Despite the lower sensitivity of heteronuclear experiments coming from the selection of 1 H-13 C isotopomers at natural abundance, these techniques are the most informative for the structure since they correlate short and long range coupled 1 H and13 C nuclei in the molecule. When the molecule is flexible, it may be important to access its 3D structure, which is possible through the Nuclear Overhauser Effect SpectroscopY (NOESY) or the Rotating frame Overhauser Effect SpectroscopY (ROESY) techniques[START_REF] Angerer | Separation and structural characterization of the new synthetic cannabinoid JWH-018 cyclohexyl methyl derivative NE-CHMIMO using flash chromatography, GC-MS, IR and NMR spectroscopy[END_REF]. For instance, those techniques can be used to determine the Z or E configuration of an alkene, the relative configuration of asymmetric carbons (R, S), while they can also provide evidence for the conformational mobility of the structure.

Figure 3 :

 3 Figure 3: NMR pulse sequences commonly used to elucidate NPS molecular structures

Figure 4 :

 4 Figure 4: NMR spectra of 3-MMC at 60 MHz and 700 MHz.

Figure 5 :

 5 Figure 5: 2D HSQC spectrum of strychnine (135 mM) dissolved in CDCl3 recorded with 128 scans in 22.7 hours at 1 Tesla. Reproduced from Ref. [34] with permission.

Figure 6 :

 6 Figure 6: Future of NPS identification.

Table 1 :

 1 UHPLC, NMR and UHPLC-MS quantitative results of MDMA tablet.[START_REF] Naqi | 1H quantitative NMR and UHPLC-MS analysis of seized MDMA/NPS mixtures and tablets from night-club venues[END_REF] -Reproduced with permission of the Royal Society of Chemistry.

	No		UHPLC		NMR		UHPLC-MS
	4 Entry	Weight (mg)	MDMA (mg) ± SD dose	RSD%	MDMA (mg) ± SD dose	RSD%	MDMA (mg) ± SD dose	RSD%
	a	512.73 160.55 ± 0.8	0.5	164.21 ± 0.4	0.3	161.07 ± 4.7	2.9
	b	481.95 150.27 ± 2.4	1.6	151.92 ± 2.1	1.4	153.80 ± 8.6	5.6


Giuseppe Gangarossa

Serge Luquet
email: serge.luquet@u-paris.fr

Hypothalamic regulation of glucose homeostasis: is the answer in the Matrix? Authors

 now reports that the integrity of arcuate perineuronal nets, an extracellular matrix component that enmesh GABAergic neurons, is reversibly altered in diabetic rats, and a key component for FGF1-mediated diabetic remission. These novel insights unravel how perineuronal nets dynamically contribute to the central control of glycemia.

Main body (998)

The brain has emerged as a prominent player in the control of feeding behavior, energy expenditure and glucose homeostasis. The arcuate nucleus (ARC) of the hypothalamus contains highly specialized neuronal populations, including AgRP-and POMC-producing neurons, which represent the two main opposite branches of the melanocortin system [START_REF] Morton | Neurobiology of food intake in health and disease[END_REF] . Several studies have consolidated the role of ARC neurons in the regulation of feeding behavior, metabolism and nutrient partitioning. Among these, recent reports have shown that a single intracerebroventricular (icv) injection of the fibroblast growth factor 1 (FGF1), a peptide produced in the brain by neurons, astrocytes and ependymal cells, leads to strikingly long-lasting normalization of blood glucose in a diabetic rodent models model [START_REF] Scarlett | Central injection of fibroblast growth factor 1 induces sustained remission of diabetic hyperglycemia in rodents[END_REF]. The ARC and the median eminence (ME) were subsequently identified as primary targets for the anti-diabetic action of FGF1 [START_REF] Brown | The Hypothalamic Arcuate Nucleus-Median Eminence Is a Target for Sustained Diabetes Remission Induced by Fibroblast Growth Factor 1[END_REF].

Taking advantage of various histological and molecular techniques in combination with in vivo metabolic analysis, Alonge and colleagues (2020) have now explored the role of perineuronal nets (PNNs) in FGF1-induced diabetic remission.

First described by Camillo Golgi more than a century ago, PNNs are specialized components of the condensed extracellular matrix that form honeycomb-like structures enmeshing the cell bodies of fast-spiking neurons, thereby controlling their activity and synaptic plasticity [START_REF] Celio | Perineuronal nets: past and present[END_REF][START_REF] Testa | Human Semaphorin 3 Variants Link Melanocortin Circuit Development and Energy Balance[END_REF]. The building blocks for PNN assembly, consisting of proteoglycans (PGs), chondroitin sulfate glycosaminoglycans (CS-GAGs) and PGs-interacting proteins, are provided by neurons, glial cells and cerebrospinal fluids to form a condensed mesh around neurons leaving space for the synaptic buttons of afferent synapses, and offering physical protection while structuring neurons plasticity and activity. PNNs-constituting GAG polysaccharides are composed of variable sequences of disaccharide units, and the tightness of the scaffolding structure surrounding neurons depends on both protein interactions and polysaccharide modifications. The appearance of PNNs coincides with the closure of the critical period during brain development and the integrity of PNNs has been involved in various brain functions and dysfunctions including memory storage, neurodegenerative and psychiatric disorders [START_REF] Testa | Human Semaphorin 3 Variants Link Melanocortin Circuit Development and Energy Balance[END_REF].

The study of Alonge and colleagues (2020) represents a converging point of several observations, mostly made by the same group. Indeed, the founding observation was that a single icv injection of FGF1 produced transient hypophagia and long-lasting (>3 weeks and up to 17 weeks) reduction in blood glucose levels in rodent models of type 2 diabetes (T2D) [START_REF] Scarlett | Central injection of fibroblast growth factor 1 induces sustained remission of diabetic hyperglycemia in rodents[END_REF].

Subsequent work reported that the postnatal formation of PNNs around ARC GABAergic neurons (mostly AgRP-neurons) and in the ME parallels the maturation of energy-sensing ARC neurocircuits in a leptin-dependent manner [START_REF] Mirzadeh | Perineuronal Net Formation during the Critical Period for Neuronal Maturation in the Hypothalamic Arcuate Nucleus[END_REF]. In adults, the hypothalamic ARC/ME was therefore identified as a primary target for the anti-diabetic action of FGF1 [START_REF] Brown | The Hypothalamic Arcuate Nucleus-Median Eminence Is a Target for Sustained Diabetes Remission Induced by Fibroblast Growth Factor 1[END_REF]. Now, Alonge and colleagues (2020) [START_REF] Alonge | Hypothalamic perineuronal net assembly is required for sustained diabetes remission induced by fibroblast growth factor 1 in rats[END_REF] reports a sharp decrease in the abundance of ARC PNNs in a hyperglycemic T2D rat model. Using a mass spectrometry technique, they highlight an imbalance in the sulfation patterns of CS disaccharide isomers toward an increased 4S/6S CS ratio that was associated with hyperglycemia in Zucker diabetic fatty (ZDF) rats compared to normoglycemic animals (Figure 1A, B), while central delivery of FGF1 resulted in a rapid (24 hrs) restoration of ARC PNNs abundance and sulfation patterns (Figure 1B). To gain insight into the function of ARC PNNs, the authors locally microinjected Chondroitinase ABC (ChABC), an enzyme which dissolves GAG into its disaccharide core units, in combination with icv FGF1 delivery in diabetic ZDF rats. While the targeted disruption of the components of ARC PNNs did not affect the anorectic responses associated with the central action of FGF1, it selectively surfeits diabetic remission. Indeed, unlike vehicle-treated diabetic rats, animals treated with ChABC experienced a transient decrease in blood glucose which rapidly returned to diabetic hyperglycemia levels. Thus, these results reveal that the integrity of ARC PNNs is necessary for the central action of FGF1 in eliciting a sustained, feeding-independent, diabetes remission (Figure 1B).

Overall, these results introduce the structure of PNNs as a new cytological architecture critically involved in the central control of energy balance and offer a yet unappreciated target for anti-diabetic strategies. However, some question remains. For instance, local ChABC application would disrupt the whole ARC PNNs cyto-structure, thereby limiting the identification of precise neural substrates.

Similarly, while the PNNs/AgRP-neurons assembly seems a reasonable target for mediating the action of FGF1, it is likely that other ARC GABAergic neurons may participate in the long-lasting regulation of blood glucose and so far, no clear molecular mechanisms connecting PNNs and ARC neurons have been identified . By the same token, ME tanycytes are gatekeepers for the entry of metabolic signals and their action on ARC neurons [START_REF] Prevot | The Versatile Tanycyte: A Hypothalamic Integrator of Reproduction and Energy Metabolism[END_REF], and they strongly respond to FGF1 [START_REF] Brown | The Hypothalamic Arcuate Nucleus-Median Eminence Is a Target for Sustained Diabetes Remission Induced by Fibroblast Growth Factor 1[END_REF]. Hence, it is tempting to speculate that the integrity of PNNs is required for tanycytes-neurons communication within the ARC/ME microenvironment, allowing for an optimized regulation of hypothalamic circuits by circulating hunger and satiety signals.

Given the role of PNNs in the maturation of hypothalamic networks [START_REF] Mirzadeh | Perineuronal Net Formation during the Critical Period for Neuronal Maturation in the Hypothalamic Arcuate Nucleus[END_REF], one would expect that any genetic or pathophysiological alteration of PNNs during development will increase the susceptibility for metabolic disorders in adulthood. Interestingly, genetic variants in the semaphorin 3A signaling pathway have recently been associated with an impaired development of hypothalamic melanocortin circuits and obesity in humans (van der Klaauw et al., 2019). As member of the chemorepulsive molecules family, semaphorin 3A is a well-known regulator of the function of PNNs (de Winter et al., 2016;[START_REF] Testa | Human Semaphorin 3 Variants Link Melanocortin Circuit Development and Energy Balance[END_REF] and, as such, could represent an interesting molecular link between ARC PNNs formation and the central control of energy homeostasis.

In conclusion, Alonge and colleagues (2020) unveil a role for PNNs in the central regulation of glucose homeostasis. Further studies are warranted to precisely ascertain the role of PNNs in the development and functions of hypothalamic metabolic circuits together with the physiological mechanisms by which ARC PNNs/neurons regulate peripheral organ activity in promoting sustained diabetic remission. Unraveling how individual components of PNNs participate in energy homeostasis may ultimately lead to novel and effective strategies against metabolic disorders.

FIGURE LEGEND

The integrity of arcuate perineuronal nets (PNNs) is essential for the control of glucose homeostasis. (A) Arcuate perineuronal nets (PNNs) enmesh GABAergic neurons, among which include AgRP-expressing neurons, and form a functional continuum with the median eminence (ME).

Core components of PNNs are hyaluronan, chondroitin sulfate (CS) CS-and dermatan sulfate (DS)-GAG glycosaminoglycan isomers (CS/DS-GAGs) and proteins interacting with proteoglycans or GAGs, such as aggrecan and tenascin (Tn-R). (B) In diabetic-hyperglycemic ZDF rats, the abundance of ARC PNNs is decreased and the proportion of disaccharides bearing sulfation in position 4 increase over that bearing sulfation in position 6 (4S/6S-CS ratio). A single intracerebroventricular injection of FGF1 results in normalization of both abundance of PNNs and corrected "sulfation code". The proper integrity of ARC PNNs is required for FGF1-mediated diabetic remission but is dispensable for the acute hypophagic response.

ACKNOWLEDGMENT

This work was supported by the Centre National de la Recherche Scientifique (CNRS), Université de Paris and La Fondation pour la Recherche Médicale (FRM). We also acknowledge the Fyssen Foundation, the Nutricia Research Foundation, the Allen Foundation Inc. and the Agence Nationale de la Recherche (ANR).

Declaration of interest

"The authors declare no competing interests"