D Mouchel

Dit Leguerrier

R Barré

Q Ruet

Daniel Imbert

C Philouze

P H Fries

V Martel-Frachet

J K Molloy

F Thomas

V Martel- Frachet

Lanthanide complexes of DOTA-nitroxide conjugates for redox imaging: spectroelectrochemistry, CEST, relaxivity, and cytotoxicity

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Introduction

Medical imaging is an extremely important domain for current research due to the necessity of precocious detection, followup, and treatment of dangerous illnesses. 1 In particular molecular imaging can provide details of biological processes in tissues in the human body, giving more information for the diagnosis of diseased tissues. 2 The development of molecular imaging requires the development of novel imaging probes capable of giving an effective response to a stimulus. The redox status of tissue is an important marker of its activity. The redox potential is defined by the sum of the redox couples weighted with the concentration of the respective reduced species. The intracellular medium is buffered by several redox couples of which the glutathione disulfide/sulfhydryl GSSG/GSH is the main one (redox potential of -120 to -70 mV). [START_REF] Jacob | Redox Signaling and Regulation in Biology and Medicine[END_REF] The redox environment of the extracellular space is dominated by the cysteine disulfide /sulfhydryl redox couple CySSCy/CysSH, with higher concentrations (0.05-0.3 mM) than the GSSG/GSH couple (0.002-0.02 mM) and with less negative redox potential (-80 to -20 mV) than GSSG/2GSH, giving a more oxidized compartment than the cytosol. [START_REF] Schafer | [END_REF] Conditions of oxidative stress are encountered when the natural metabolism of oxygen is disrupted, leading to the over production of reactive oxygen species (ROS) by incomplete reduction. 5 Changes in redox status (oxidative stress or hypoxia) are believed to contribute to cellular signalling (differentiation, apoptosis), [START_REF] Hurd | Redox Signaling and Regulation in Biology and Medicine[END_REF] but also the proliferation of diseases such as cardiovascular diseases, 7, 8 cancers 9,10 or neurodegenerative diseases 11 . Among various non-invasive detection techniques, magnetic resonance imaging (MRI) has proved to be a key technique for clinical imaging. It relies on the differential relaxation of water protons of the bulk water in the human body. [START_REF] Helm | Contrast Agents for MRI: Experimental Methods[END_REF] Its success lies in its high spatial resolution, lack of hazardous radiation, and the fact it is painless and can be functional in addition to anatomical. Approximately one third of the MRI scans are realized with a relaxation contrast agent (CA), which is often a gadolinium complex. Gd III is the metal ion of choice due to its quenched total electronic orbital angular momentum L = 0, the highest possible total electronic spin value S = 7/2, and a slow longitudinal electronic relaxation, especially at the rather high magnetic field B0 ≥ 1 T of the MRI scanners. [13][14][15] The CA is injected into a vein before the analysis to improve the details and clarity of the images. The CA effectiveness is gauged by its relaxivity r1, which is the increase in the longitudinal relaxation rate of the water protons (hydrogen nuclei) per mM of contrast agent in biological tissue. 11,14 Recently, Chemical Exchange Saturation Transfer (CEST), another technique of MRI, based on paramagnetic lanthanide ions such as Eu III , Yb III , and Dy III with very fast electronic relaxation lends itself to more sensitive specific functional imaging. It was suggested almost ten years ago that lanthanide-based CAs could be also used to sense the redox environment. 16,17 Two main strategies emerged, which are based on either a redox-reactive ligand or redox-active metal. In the first case the r1 value of the Gd III -based CA can be tuned by the hydration state (number of coordinated water molecules q), which is itself modulated by a redox event at the ligand. This first strategy is illustrated by DOTA-based Gd 3+ complexes (DOTA is 1,4,7,10-Tetraazacyclododecane-1,4,7,10tetraacetic acid) appended by a merocyanine group. This group cyclizes into spyrooxazine upon reduction by NADH, resulting in decoordination and further increase of q from 1 to 2 with change in r1. [18][19][20] Several Gd 3+ complexes functionalized by a thiol-reactive 2-pyridyldithio group were also prepared and shown to react with GSH with a change in q number from 2 to 1. 21,22 Alternatively, the slower rotational dynamics upon redox reaction of the ligand by reversible (or irreversible) binding of low molecular weight Gd 3+ complexes to macromolecules can be used to increase r1 values at constant q. Some Gd 3+ -DOTA complexes appended by terminal thiol groups demonstrated such behaviour, whereby they react with the Cys-34 residue of human serum albumin. 23 The second strategy is exemplified by cryptates of europium, the only lanthanide susceptible to adopt two different oxidation states in vivo. 24 The Eu 2+ ion, isoelectronic to Gd 3+ , is oxidized in vivo into Eu 3+ , which shows a negligible paramagnetic relaxation because of its very fast electronic relaxation. [25][26][27] Chart 1. Structures of ligands whose Eu 3+ complexes were reported as redox-responsive PARACEST agents. 28 Paramagnetic Chemical Exchange Saturation Transfer (PARACEST) is an attractive contrast mechanism for MRI, which often employs paramagnetic lanthanide complexes. 29 A presaturation pulse is applied to exchangeable protons within the complex, resulting in a transfer of saturated spins to the bulk. As a consequence, the water signal intensity decreases and negative contrast is observed. Very few lanthanide redoxresponding complexes were reported to date in PARACEST imaging. 16 All are based on Eu 3+ complexes of DOTA tetraamide ligands as this lanthanide shows the slowest water exchange rates (Chart 1), 28 and operate according to two distinct mechanisms: The ligand can undergo reduction such as in a nitrobenzene (Chart 1A) 30 or quinolinium group, 31 resulting in an alteration of the water exchange rate. Alternatively, the nitroxide appended Eu 3+ DOTA complex (Chart 1B) exhibits a T1shortening of bulk water protons by the paramagnetic centers, which increases upon reduction. 32 Interestingly a Eu 3+ /Eu 2+ switch was recently proposed as a redox PARACEST agent. 33 We recently reported Ln III complexes, which are sensitive to environmental redox changes. We monitored the change in oxidation state by luminescence, where over 90 % switch could be achieved. [34][35][36] We have also demonstrated through EPR the spin trap of a major ROS, the hydroxyl radicals, by lanthanide complexes of a DOTA-hydroxylamine derivative. 37

Chart 2. Structures of ligands L1 and L2

Following the elegant strategy developed by Sherry et al. to design PARACEST agents responsive to redox changes 32 we herein report a series of lanthanide(III) complexes (Gd, Eu, Dy, Yb) based on a cyclen scaffold containing the redox active TEMPO arm 38 (Chart 2). Two different derivatives L1 and L2 have been designed containing three acetamides or with an alkyne. The alkyne was chosen in order to allow future functionalization and bioconjugation of these complexes via click chemistry. We investigate their photophysical, redox, relaxometry and their potential as redox-responsive PARACEST agents.

Results and discussion

Synthesis

The synthesis of ligands L1 and L2 was performed using the successive functionalization of the cyclen macrocycle (scheme 1). L1 was synthesised via substitution with the nitroxide acetamide and further functionalization with three bromoacetamide arms. L2 was synthesised by protection of the cyclen in order to selectively functionalise 2-bromoacetamide arms, deprotection, followed by the addition of a dilute solution of propargyl bromide to perform the mono substitution. Purification and further addition of the nitroxide acetamide afforded the desired product (see experimental). The Gd III , Eu III , Yb III and Dy III complexes were prepared from L1 and L2 by reaction with the corresponding lanthanide salt in H2O at pH 7.4 and precipitation of the resulting complex. Ligand L2 was crystallised from slow evaporation of water solution to give single crystals of quality for X-Ray diffraction. Surprisingly, structural analysis demonstrated the complexation of a Na + ion in the cyclen cavity, giving the species [Na(L2)] + , 0.5 Br -, 0.5 Cl -. The sodium ion is heptacoordinated to four nitrogens of the macrocycle and three oxygens of the acetamide arms (Fig. 1). The Na-N bond distances range between 2.527(10) and 2.643(9) Å, while the Na-O are in between 2.398(3) and 2.436(3) Å. The C2-C3 bond distance in one pending arm is 1.17(2) Å, which confirms its triple bond character. The N-O bond distance in the opposite pending arm is 1.289(4) Å, i.e. reminiscent of TEMPO radicals.

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Electrochemistry

Electrochemical investigations were performed by cyclic voltammetry (CV), differential pulse voltammetry (DPV) and rotating disc electrode (RDE) voltammetry in an acetonitrile solution containing 0.1 M tetra-n-butylammonium perchlorate (TBAP) as supporting electrolyte. All the potentials are referred to the ferrocenium/ferrocene redox standard. The ligands demonstrate oxidation potentials of 0.28 V and 0.31 V vs. Fc + /Fc (0.91 to 0.94 V vs. NHE) 39 respectively for L1 and L2. The complexes exhibit a reversible oxidation wave in the range 0.26 -0.34 V vs. Fc + /Fc (Table 1, Fig. 2), which is assigned to the oxidation of the TEMPO moiety into the persistent oxoammonium species TEMPO + . 40 These values are slightly more positive than that reported for free TEMPO (0.21 V vs. Fc + /Fc) 41 due to the presence of the amide function. An irreversible reduction wave corresponding to the reduction of the TEMPO into the hydroxylamine TEMPOH is observable as a broad feature between -1 and -1.5 V vs. Fc + /Fc (about -0.3 to -0.9 V vs NHE, Fig. 2). 40 The apparent irreversibility of the redox wave is due to a very slow electron transfer for the protoncoupled TEMPOH/TEMPO redox couple and an exceedingly large anodic-to-cathodic peak separation, which shifts the oxidation peak of TEMPOH above the TEMPO + /TEMPO wave. 40,42 Each complex, Eu III , Dy III , Gd III or Yb III demonstrates similar features with only slight shift of the oxidation and reduction waves, confirming that the metal ion had very little effect on the redox behaviour of the TEMPO group. It must be stressed that no oxidation or reduction of the Ln III centre was observed in the investigated potential range.

Absorption and luminescence spectroscopy

The UV-Vis absorption spectra of the ligands L1 and L2 in CH3CN show two main bands: 246 nm (ε = 14 200 M -1 cm -1 ) and 286 nm (345 M -1 cm -1 ) for L1 and 246 nm (22 530 M -1 cm -1 ) and 286 nm (1590 M -1 cm -1 ) for L2. [START_REF]The difference in molar absortivity was not due to distinct oxidation state or purity of the samples[END_REF] Upon complexation, these bands were slightly red shifted with respect to the ligand, with the [Gd(L2)] 3+ complex showing λ of 292 nm (4020 M -1 cm -1 ) and 363 nm (ε = 1760 M -1 cm -1 ). The complexes also possess very weak epsilon absorption corresponding to the f-f lanthanide transitions. The luminescence spectra were recorded for the europium complexes since structural information can be extracted from a 7 F0-5 band shape analysis. They were recorded via excitation in the ligand at 315 nm providing the expected spectra of the Eu III ion (Fig. 3). A sharp band is observed at 580 nm for [Eu(L1)] 3+ that corresponds to the transition 5 D0 → 7 F0. Its sharpness suggests high symmetry in the coordination sphere of the complex and presence of single site symmetry for Eu III . The 5 D0 Table 2. UV/Vis data of the nitroxide complexes [a] Complex  (nm) / (M -1 cm -1 ) [Gd(L1)] 3+ 245 ( 30 [a] In 0.5 mM CH3CN solutions containing TBAP as supporting electrolyte (0.1 M). T = 298 K. In order to ensure full nitroxide radical formation the complexes were subjected to electrolysis at a potential intermediate between the TEMPOH/TEMPO and TEMPO + /TEMPO redox couples.

Luminescence measurements were also used to determine the lifetimes of the complexes via excitation at 396 nm in both H2O and D2O. For [Eu(L1)] 3+ we determined t=0.43 s in H2O and t=1.32 in D2O, representing one water molecule in the coordination sphere of the Ln III ion. This result is mostly as expected due to the potential eight coordinate macrocyclic ligand. Complex [Eu(L2)] 3+ demonstrates t=0.44 s and t=1.36 s for H2O and D2O, respectively, again calculating one coordinated water molecule. Hence the coordination number evolves upon substitution of one amide with the alkyne, but the number of coordinated water remains constant, q = 1. This water molecule is crucial in the use of these complexes in imaging.

Electron Paramagnetic Resonance spectroscopy

Both the lanthanide ions and the ligands harbour unpaired electrons and hence can be analysed by electron paramagnetic resonance (EPR).

We first recorded the fluid solution spectra of the free ligands L1 and L2 at T = 293 K. They show a typical isotropic 3-line pattern centered around g = 2.006 (Fig. 4). The hyperfine coupling constant AN is 1.7 mT, typical for TEMPO radicals. In the absence of metal ions the broadening of the high field component is solely due to the Brownian rotational motion of the radical. In the fast tumbling regime, the rotational correlation time c can be calculated by using the Kivelson's formula (eq. 1), where C is a constant calculated from the principal values of g and A tensors of the nitroxide radical (6.6 10 -10 for for di-tertbutylnitroxide -this value is not expected to change much between nitroxide radicals), [START_REF] Libertini | [END_REF]45 H(+1) is the peak-to-peak linewidth of the low field line and h(+1) and h(-1) are the height of the low and high field resonances.

c = C H(+1)[(h(+1)/h(-1)) 1/2 -1]
(Eq. 1) 3+ and [Gd(L2)] 3+ at T = 293 K show noticeable differences. Both display the sharp 3-line nitroxide pattern ( = 0.28 mT) with weaker intensity in comparison to the free ligands. In the case of [Gd(L1)] 3+ this 3-line pattern is overlapped with an intense and broad resonance ( = 3.24 mT) centered at g = 1.99, which is assigned to the gadolinium signal (Fig. 4). Upon addition of ascorbic acid the TEMPO unit is reduced to TEMPOH, resulting in a quenching of the 3-line pattern without affecting the broad resonance. Strikingly, the gadolinium resonance cannot be observed in the case of [Gd(L2)] 3+ . This suggests faster relaxation and hence distinct influence of the nitroxide and coordination environment on the gadolinium relaxation rate.

We also investigated the reversibility of the reduction by adding the oxidant NaIO4 to an ascorbate-reduced sample of [Gd(L2)] 3+ (Fig. S15). The initial radical signal of [Gd(L2)] 3+ was restored to more than 80 % after 30 min. The ascorbate-reduced sample could be alternatively reoxidized by O2 bubbling in basic conditions, but the reaction proceeds less efficiently (only 20 % recovery after 30 min). Low temperature measurements were conducted (4 -50 K) in order to characterize the Yb, Eu and Dy complexes, wherein fast relaxation prevents observation of the lanthanide at higher temperatures. At T = 40 K the complexes exhibit the typical signature of immobilized nitroxide radicals, with broadening and shift of the mI = +1 and -1 transitions due to anisotropy. Accordingly, similar spectra were obtained for the free ligands in frozen solution (see ESI). Upon cooling down to 5 K, drastic changes were evidenced in the EPR spectra for [Yb(L1)] 3+ , [Dy(L1)] 3+ and their alkyne counterparts. For a given Ln ion the EPR spectra of the alkyne derivatives [Ln(L2)] 3+ show the same temperature dependence than [Ln(L1)] 3+ and hence will not be commented on detail. The spectrum of [Yb(L1)] 3+ taken at 4 K shows an intense doublet at g = 2.00 (Fig. 5), which corresponds to a radical signature but differs markedly from the 3-line pattern of isolated nitroxides. The spectrum of the free ligand L1 was indeed recorded at this low temperature and shows exclusively the anisotropic 3-line pattern of the nitroxide radical. Hence this behaviour strongly suggests that the paramagnetic lanthanide interacts magnetically with the nitroxide moiety. From the X-Ray crystal structure of NaL1 + the N(nitroxide)-metal distance could be estimated at ca. 7.8 Å, confirming that a triplet state may be accessible. Considering the feature at 4 K as a spin triplet resonance, and using a simple model with an isotropic g tensor the simulation gives |D| = 300 MHz, which is consistent with the above interspin distance. Note that simulation of this feature by considering an isolated nitroxide anisotropic signal leads to unreasonable 14 N hyperfine coupling constants. The addition of ascorbate to [Yb(L1)] 3+ results in the full quenching of this feature around g = 2, confirming its assignment. The EPR spectrum of the reduced [Yb(L1)] 3+ shows resonances at g = 8.6, 6.41 and 5.2, which are assigned to the parallel features of an isolated Yb 3+ ion (Fig. 6). The main line at g = 6.41 is assigned to the even isotope, while the satellite ones (approx. 15 % in intensity) correspond to the 171 Yb 3+ and 173 Yb 3+ isotopes. The g// value of the even isotope compares well with that reported for the [Yb⋅DTMA⋅OH2] 3+ complex (6.45), 46 congruent with a similar environment of the metal center.

For [Dy(L1)] 3+ the sharp nitroxide lines decrease in intensity upon decreasing the temperature, at the expense of a broader signal centered around g = 2 that we assign to the interacting nitroxide, as observed for [Yb(L1)] 3+ . It is distributed over a larger window in comparison to [Yb(L1)] 3+ , indicative of a distinct influence of the lanthanide ion on the radical moiety. This is also revealed by the spectrum at T = 8K, which still contains both the sharp nitroxide lines and the broader line (see ESI). After addition of ascorbate (reduction of the nitroxide unit) the signal at g = 2 completely disappears, affording a spectrum with an intense resonance at geff ≈ 11.6, which is reminiscent of isolated Dy 3+ complexes (Fig. 6).

The EPR spectrum of [Eu(L1)] 3+ at 40 K shows the same nitroxide feature as [Yb(L1)] 3+ and [Dy(L1)] 3+ . At 4 K the nitroxide signal broadens slightly, but does not show any distinct evolution of its shape, which is likely the consequence of the small magnetic moment of trivalent europium. Please do not adjust margins Please do not adjust margins

DFT calculations on model compounds

The structure of the lanthanide complexes was investigated by DFT calculations. In order to overcome the prohibitive computational cost on complexes of f-metal ions the optimizations were performed on two models wherein the lanthanide ion was replaced by Y 3+ , namely YL1 3+ and YL2 3+ . The yttrium is indeed a common substitute of Gd 3+ in imaging. The number of coordinating water molecules was set to one, in agreement with the luminescence data (see above). For YL1 3+ the optimized structure shows a pendent TEMPO arm, with the hydrogens of the water molecule pointing along but opposite to the TEMPO group. For YL2 3+ we first performed a relaxed potential energy surface (PES) scan along the C-N axis of the alkyne arm to determine its preferred orientation. The lowest energy conformation corresponds to the alkyne pointing away from to the metal. It also interesting to note that the water hydrogens point orthogonal to the alkyne and establish Hbonds with the two acetamide arms. Hence this conformation allows minimization of the steric clash between the coordinating water molecule and the alkyne group, while stabilizing YL2 3+ through H-bonding interactions. The distinct orientation of the water hydrogens in YL1 3+ and YL2 3+ prompted us to perform an additional relaxed PES scan, whereby the water molecule was rotated along the M-O axis ( angle in Fig. 7). For YL1 3+ the variation of energy is monotonic and sinusoidal with alternating minima and maxima every ca. 120° and a total change that does not exceed 0.3 kcal/mol. Model YL2 3+ exhibits a strikingly different behaviour: the total energy change during the scan is one order of magnitude higher, with the lowest energy conformation corresponding to the water hydrogens orthogonal to the alkyne. These results show that the barrier for the rotation of the water molecule is very small for YL1 3+ but significantly higher (immobilized) in YL2 3+ . The functionalization of the macrocycle via an amide linker (TEMPO arm) hence appears to weakly affect the rotational dynamic, in contrast with alkyl linkers (alkyne arm), which do not allow for the formation of stabilizing H-bonds with the coordinated water and prevent rotation. Furthermore the coordination bonds are shorter in YL2 3+ , which is not surprising owing to the lower expected coordination number in this complex.  is the dihedral angle between one water hydrogen, the water oxygen, yttrium and the O2 oxygen of the ligand (amide connecting the TEMPO arm). The O1, O3 and O4 oxygens are from carboxylate groups.

CEST activity and relaxivity

The ligand design incorporated exchangeable NH2 protons, which can confer potential CEST activity. In addition, these complexes are 8 or 9-coordinate, thus containing a metal bound water molecule that can confer CEST activity via an exchangeable H2O on the metal centre. CEST spectra, plotted as a percentage decrease in water proton magnetisation as a function of the presaturation pulse frequency, were recorded in HEPES buffer 0.1 M in H2O:acetonitrile (2:1) at 500 MHz on both the nitroxide and hydroxylamine forms of complexes [Ln(L1)] 3+ and [Ln(L2)] 3+ (Ln = Eu III , Dy III , Yb III ). The activity of [Eu(L1)] 3+ in its TEMPO form shows a weak CEST spectrum (6% in intensity, Fig. 8) as the relaxation of the water protons neighbouring the TEMPO unit is accelerated by the magnetic moment of the free electron spin of that unit. 32 Upon addition of ascorbic acid the TEMPO unit is reduced, affording a Please do not adjust margins Please do not adjust margins closed-shell ligand. There is no more acceleration of the relaxation of the vicinal water protons. As a consequence the complex [Eu(L1)] 3+ in the reduced form demonstrated a significant CEST peak at + 51 ppm with respect to the bulk water proton resonance. The exchangeable water molecule on the metal centre presents a larger CEST effect of 12 %. The CEST effect demonstrates small pH dependence with values varying from 9 % to more than 13 % between pH 5 and 8. This small pH dependence demonstrates that the metal bound water molecule is little affected by pH changes. The CEST effect in [Eu(L1)] 3+ is within the same range, albeit slightly weaker, than for the Eu 3+ complex of the ligand depicted in Chart 1b in aqueous medium.

Relaxometry

The slow electronic relaxation of the f 7 Gd III ion makes this metal ion inefficient as PARACEST agent, but ensures its importance as relaxation CA. The relaxivity r1 has been studied by Fast Field Cycling (FFC)-NMR. It shows interesting behaviour. At high field (30MHz), [Gd(L1)] 3+ provides a T1 relaxation time of 0.26s corresponding to a r1 of 1.92 mM -1 .s -1 and [Gd(L2)] 3+ yields a T1 relaxation time of 0.16s corresponding to a r1 of 3.18 mM -1 .s -1 .

A very small change between the oxidised and reduced species of 2% and 4% in relaxivity was observed for [Gd(L1)] 3+ and [Gd(L2)] 3+ , respectively. At low field (30kHz), [Gd(L1)] 3+ provides a T1 relaxation time of 0.15s corresponding to a r1 of 3.33 mM -1 .s -1 and [Gd(L2)] 3+ yields a T1 relaxation time of 0.09s corresponding to a r1 of 5.28 mM - 1 .s -1 . Upon reduction of nitroxide into hydroxylamine, the relaxivity decreases by 13% and 44% for [Gd(L1)] 3+ and [Gd(L2)] 3+ , respectively. In the high-field region, it is known that low exchange rate of the water molecules in the first coordination sphere and fast rotational correlation time of the complex limit the relaxivity of a Gd III -based CA. Besides, in the low-field region, fast electronic relaxation limits the relaxivity under 0.1 T (3-4 MHz). 47,[13][14][15] High relaxivity values in this region are typical of highly symmetric Gd III complexes with long electronic relaxation times. 48 Complex [Gd(L1)] 3+ exhibits a higher symmetry than [Gd(L2)] 3+ and consequently presents a longer electronic relaxation time, allowing visualization of the Gadolinium signal in the EPR spectrum (0.34 T). Surprisingly, [Gd(L2)] 3+ in its reduced form shows similar relaxivity as [Gd(L1)] 3+ at low field, but significantly higher value at high field. Much more research beyond the objectives of this article is yet needed to unravel the physical origins of the observed effects. 14 It is worth noting that the influence of the redox status on the relaxivity is weak for [Gd(L1)] 3+ , ranging from 20% increase in the low field region to less than 5% in the high field region upon oxidation of the hydroxylamine moiety. Complex [Gd(L2)] 3+ shows a better response to the redox stimulus with up to 75% increase in the low field region upon hydroxylamine to nitroxide conversion. At the highest field the difference in relaxivity between both complex oxidation state remains very weak. This journal is © The Royal Society of Chemistry 20xx

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The T1 relaxation time of the water protons is modulated by the redox state without influencing the exchange rate value as demonstrated by D. Sherry and coworkers. 32 This is an interesting trend whereby the formation of a paramagnetic radical center in the vicinity of the Gd 3+ alters significantly the relaxation of the protons of the coordinated water. Hence complexes [Gd(L1)] 3+ and [Gd(L2)] 3+ could be considered as gadolinated redox probes though the relaxivity values are still too low to envisage their use as functional MRI probes.

Biological studies

The series demonstrate slight toxicity above 1 M, with invariably 60-70% of cell viability for treatments with up to 40 M of the compounds. The difference of toxicity is clearly inferred to the substitution of one acetamide arm by the alkyne group, but it is yet unclear why this modification has such an effect. One possible explanation for the absence of toxicity of the [Ln(L1)] 3+ series is a lack of cellular penetration, in line with clinically approved MRI CAs, which are injected in the circulating system. We quantified the cellular penetration of one representative complex [Dy(L1)] 3+ by using EPR spectroscopy.

The complex was incubated with M21 cells at 200 µM, which was a minimal concentration for straightforward detection. The supernatant, cell pellets and cell lysate were next analysed by EPR and the intensity of the nitroxide lines was quantified under non-saturating conditions. No signal was detected within the detection limit of EPR (<0.1% penetration) in either the cell pellets or the cell lysate. The complex was found to be localized in the supernatant, as demonstrated by a similar integration of the nitroxide resonances of the supernatant and a blank containing the complex at similar concentration in a culture medium lacking cells. Both the polar nature of the complex and its tripositive nature most likely prevent cell penetration, which may be an advantage for use as MRI agent. It must be stressed that the alkyne moiety does not affect the cell penetration, but offers a possibility for vectorising the complex by adequate functionalization. Finally, the complexes show only weak or no toxicity at all at concentrations up to 40 M against cells, which is inferred to a cellular penetration. These results demonstrate that the original concept of redox-active lanthanide complexes has large potentialities for biological imaging (CEST, MRI). Further works are currently in progress in our laboratory to increase the CEST effect and incorporate sensitizing units for bimodal magnetic/optical imaging.

Experimental Section 4.1 Materials and methods

All chemicals were of reagent grade and were used without purification. High resolution mass spectra were recorded on a Waters Xevo G2-S QTof apparatus. The UV/Vis spectra were recorded on a Cary Varian 50 spectrophotometer. NMR spectra were recorded on a Bruker AM 400 ( 1 H at 400 MHz) spectrometer. Chemical shifts are quoted relative to tetramethylsilane (TMS). Xband EPR spectra were recorded on a Bruker EMX Plus spectrometer equipped with a Bruker Helium flow cryostat and a dual mode cavity. The relaxivity values r1 were derived from the experimental longitudinal relaxation times T1 of the water protons. 14 The T1 values were measured from 20 kHz to 30 MHz with a commercial Spinmaster FFC 2000 Stelar relaxometer 49,50 Please do not adjust margins experiments. For each magnetic field r1 was calculated according to r1 = (R1-R10)/[complex], where R1 is the relaxation rate measured in the presence of the complex, R10 the relaxation rate measured in the absence of complex (typically 0.4 s- 1) . The complex concentration is expressed in mM. Cyclic voltammetry curves as well as differential pulse voltammetry curves were recorded using a CHI 620 potentiostat. The measurements were performed in 0.5 mM CH3CN solution containing 0.1 M tetra-n-butyl ammonium perchlorate (TBAP) as supporting electrolyte. Experiments were performed in a standard three-electrode cell under argon atmosphere. A glassy carbon disc electrode (3 mm diameter), which was polished with 1 mm diamond paste, was used as the working electrode. The auxiliary electrode is a compartmentalized platinum wire, while an Ag/AgNO3 0.01 M electrode was used as reference.

Luminescence spectra of the lanthanide complexes were recorded using a modular Fluorolog FL3-22 spectrometer Horiba-Jobin Yvon-Spex equipped with a double grating excitation monochromator and an iHR320 imaging spectrometer. Hamamatsu R928P and Hamamatsu R5509 photomultipliers were used for visible and NIR measurements, respectively. All spectra were corrected for detection and optical spectral response (instrumental functions) of the spectrofluorimeters. Quartz capillaries 4 mm in diameter were used. For the acquisition of the excitation and emission spectra in the NIR, a long pass coloured filter was always used at 870 nm to block the signal of the 2nd harmonics. The q value was determined by using the formula q = 1.2 [Δkobs -0.25 -0.075q CONHR ], 51 which gives for the [Ln(L1)] 3+ series q = 1.2 [Δkobs -0.55] and for the [Ln(L2)] 3+ series q = 1.2 [Δkobs -0.48].

Synthesis

4-Amino-2,2,6,6-tetramethylpiperidine-1-oxyl, free radical was purchased from Sigma-Aldrich; 1,4,7,10-Tetraazacyclododecane-1,7-bisacetamide (2) was synthesized according to literature procedures 52 .

4-(2-chloroacetamido)-2',2',6',6,'-tetramethylpiperidine-1'oxyl, free radical (0)

To a solution of 4-amino-tempo, free radical (0.200 g, 1.17 mmol) in CH2Cl2 (7 mL) was added Et3N (310 µL, 2.2 mmol, 1.9 eq). Chloroacetyl chloride (140 µL, 1.8 mmol, 1.5 eq) in CH2Cl2 (5 mL) was added dropwise at 0°C. After the addition was complete, the reaction mixture was allowed to warm to room temperature and stir for 6 hours. The reaction mixture was quenched with 3 mL of water. The organic phase was washed with HCl (1M) (2x10 mL) and NaHCO3 sat (2x20 mL). The organic phase was dried using MgSO4 and the solvent was removed under reduced pressure. The product was purified by column chromatography (CH2Cl2/MeOH; 95/5; v/v). The solvent was removed under reduced pressure to obtain the title compound as a red solid (85%). MS-ESI m/z [M+H] + : 247.08. Under argon atmosphere, to a solution of cyclen glyoxal (0.536 g, 2.8 mmol) in THF (10 mL) was added 0 (0,750 g, 3.0 mmol, 1.1 eq) in THF (5 mL). The reaction mixture was stirred at room temperature for 24 hours. A precipitate was formed, isolated by filtration and washed with diethyl ether. The resulting solid was dissolved in THF (20 mL) and o-phenylenediamine (0.108 g, 1.0 mmol, 1.1 eq) was added. The mixture was stirred at room temperature for 7 days. The residue was then filtrated and rinced with diethyl ether and the desired product was obtained in 33% yield. 1 H NMR (400 MHz, MeOD) δ = 3.10-2.80 (m br, 16H), 1.80 (d br, J = 12.5 Hz, 2H), 1.46 (t, J = 13.3 Hz, 2H), 1.17 (d, J = 5.6 Hz, 12H); 13 The monosubstituted cyclen 1 (0,334 g, 0.87 mmol, 1eq) was dissolved in 20 mL of MeCN. 2-bromoacetamide (0.419 g, 3.0 mmol, 3.5 eq), potassium carbonate (0.420 g, 3,0 mmol, 3.5 eq) and potassium iodide (0.540 g, 3.3 mmol, 3.8 eq) was added. The reaction was stirred at 50°C for 5 days. The solution was filtrated and the filtrate was evaporated under reduced pressure. The product was purified by an alumina column chromatography (EtOH/NH4OH) to afford the title compound as an orange solid (0.587 g, 99%). 1

General procedure for the synthesis of the complexes [Ln(L1)] 3+ :

The ligand L1 was dissolved in water (10 mL) and the lanthanide salt was added to the solution. The pH was adjusted to 7 by the addition of sodium hydroxide (1 M). The reaction mixture was stirred and heated to 50°C for 1 day. Water was evaporated in reduced pressure. The yellow solid was dissolved in minimum of methanol and precipitated in diethyl ether. It was then dissolved in acetonitrile, filtrated and solvent was evaporated to give the desired compound [Ln(L1)] 3+ as an orange powder. [Yb(L1)] 3+ : from L1 (100 mg, 0.15 mmol) and ytterbium triflate (93 mg, 0.15 mmol). Yield: 100%. 1 To a solution of 2 (1.31 g, 4.6 mmol) and NaOAc (0.804 g, 9.9 mmol, 1.2 eq) in EtOH (200 mL) was cooling at 0°C. Propargyl bromide (580 µL, 5.2 mmol, 1.2 eq) in EtOH (100 mL) was added dropwise at 0°C. After the addition was complete, the reaction mixture was allowed to stir for 4 days at 50°C. The solvent was removed under reduced pressure. The residue was taken up in MeCN and filtered. The solvent was removed under reduced pressure to obtain the title compound as a brown oil (93%). 1 To a solution of 3 (1.39 g, 4.3 mmol, 1 eq) in EtOH (100 mL), K2CO3 (1.18 g, 8.6 mmol, 2 eq), KI (1.46 g, 8.8 mmol, 2.1 eq) and 1 (1.18 g, 4.8 mmol, 1.1 eq) was added. The reaction mixture was stirred for 5 days at 60°C. The solution was filtered on celite, rinsed EtOH and the solvent was removed under reduced pressure. The product was purified by an alumina column chromatography (AcOEt then EtOH/NH4OH). The solvent was removed under reduced pressure to obtain the title compound as an orange solid (88 %). Slow evaporation of water led to orange crystals which were determine by X-ray crystallography. Reduction with sodium ascorbate was used to characterized the ligand. 1 H NMR (400 MHz, D2O) δ = 4.53 (s, 2H), 4.22 (t, J = 10.5 Hz, 1H), 3.56 (br s, 8H), 3.02 (s, 16H), 1.94 (d, J = 13.0 Hz, 2H), 1.60 (t, J = 13.0 Hz, 2H), 1.28 (d, J = 16.4 Hz, 12H); 13 C NMR (100 MHz, D2O) δ = 78. 4, 69.6, 62.5, 43.1, 29.9, 19.6; IR: 3360, 3283, 3194, 3123, 2977, 2941, 2825, 1653, 1568, 1456, 1363, 1320, 1108, 647; HRMS (m/z): [M+H] + calcd for C26H48N8O4, 536.379; found, 536.379.

General procedure for the synthesis of the complexes [Ln(L2)] 3+ :

The ligand L2 was dissolved in water (20 mL) and the lanthanide salt was added to the solution. The pH was adjusted to 6.5 by the addition of sodium hydroxide (1 M). The reaction mixture was stirred and heated to 50°C for 2 days. Water was evaporated in reduced pressure. Purification by precipitation in diethyl ether gives the desired compound [Ln(L2)] 3+ as an orange powder. [Eu(L2)] 3+ : from L2 (300 mg, 0.45 mmol) and europium triflate (272 mg, 0.45 mmol). Yield: 83%. 1 H NMR (400 MHz, MeOD) δ = 18.1, -2.1, -6.3, -7.3, -9.0, -10.9, -12.7, -17.0; IR (cm

X-Ray diffraction.

Single crystals were coated with perfluoropolyether, picked up with nylon loops and mounted in the nitrogen cold stream of the diffractometer. Mo-Kα radiation (λ = 0.71073Å) from a Mo-target rotating-anode X-ray source equipped with INCOATEC Helios mirror optics was used. Final cell constants were obtained from least squares fits of several thousand strong reflections. Intensity data were corrected for absorption using intensities of redundant reflections with the program SADABS. The structures were solved by Patterson methods and subsequent difference Fourier techniques. The OLEX software was used for the refinement. 53 All non-hydrogen atoms were anisotropically refined and hydrogen atoms were placed at calculated positions and refined as riding atoms with isotropic displacement parameters. CCDC-1997062 contain the crystallographic data for [Na(L2)] + (Cl -); these data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html.

DFT Calculations

Full geometric optimizations were performed with the Gaussian 9.0 program. [START_REF] Frisch | Gaussian 09, Revision D.01[END_REF] The B3LYP functional [START_REF] Lee | [END_REF]56 was used together with the 6-31g* basis set for the C,H,N atoms 57 and a pseudo potential (LanL2DZ) 58 for the central metal ion. Frequency calculations were systematically performed on the optimized structures in order to ensure that they correspond to a real energy minimum and not a saddle point. The relaxed potential energy surface scans were performed by varying dihedral angles.

Cell culture

Human melanoma cell line M21 was purchased from ATCC (Molshein, France). Cells were cultured in DMEM medium supplemented with 10% (v/v) fetal calf serum (FCS) and 2 mM glutamine (Thermo Fisher Scientific, Courtaboeuf, France). Cells were maintained at 37 °C in a 5% CO2-humidified atmosphere and tested to ensure freedom from mycoplasma contamination. All cell lines were used within 5-50 passages of thawing the original stocks.

Scheme 1 .

 1 Scheme 1. Synthetic procedures towards L1, L2 and their lanthanide complexes a a Reaction conditions: (i) chloroacetyl chloride, Et3N, 0°C to rt, CH2Cl2, overnight; (ii) 1) 0, THF, 1 day, 2) o-PDA, MeOH, 1 week; (iii) 2-bromoacetamide, K2CO3, KI, MeCN, 50°C, 5 days; (iv) Ln salt, H2O, pH 7, 50°C, overnight; (v) 1) 2bromoacetamide, MeCN, 1 day, 2) o-PDA, MeOH, overnight, 3) CsCO3, MeOH, Overnight; (vi) 2-propargyl bromide, NaOAc, EtOH, 0 to 50°C, 4 days; (vii) 0, K2CO3, KI, EtOH, 60°C, 5 days; (vii) Ln salt, H2O, pH 6.5, 50°C, 2 days.

Figure 1 .

 1 Figure 1. ORTEP plot of [Na(L2)] + (the H atoms are omitted for clarity)

  [a] In 0.5 mM CH3CN solutions containing TBAP as supporting electrolyte. All the potentials values are given in V and referred to the Fc + /Fc redox couple. T = 298 K; scan rate, 0.1 V/sec. Parameters for the ferrocene against the reference used (AgNO3 0.01 M): E1/2 = 0.090 V. In order to convert the potentials from the Fc + /Fc reference to the NHE 0.630 V must be added to the listed values.39

Figure 2

 2 Figure 2 Cyclic voltammetry curve [Eu(L2)] 3+ in 0.5 mM CH3CN solution containing 0.1 M TBAP. Scan rate, 0.1 V s -1 , T = 298 K, carbon electrode.

<

  This journal is © The Royal Society of Chemistry 20xxPlease do not adjust margins Please do not adjust margins → 7 F1 transition is the magnetic dipole transition independent of the coordination sphere. The hypersensitive electric dipole transition 5 D0 → 7 F2 is the most influenced by the local symmetry. The distinct ratio of the integrated bands corresponding to the transitions 5 D0 → 7 F2 over 5 D0 → 7 F1 in [Eu(L1)] 3+ and [Eu(L2)] 3+ suggests a different coordination environment. Most particularly the higher ratio in [Eu(L2)] 3+ (Fig.3) supports a lower symmetry around the first coordination sphere. Finally, excitation spectra confirmed the excitation occurred via population of ligand and transfer towards the Eu III metal.

Figure 3

 3 Figure 3 Luminescence spectra of [Eu(L1)] 3+ (left) and [Eu(L2)] 3+ (right) in D2O upon excitation at 395 nm.

  040), 292 (5770), 363 (3000) [Eu(L1)] 3+ 245 (26 970), 292 (8110), 363 (4270) [Dy(L1)] 3+ 245 (34 190), 292 (3620), 363 (1800) [Yb(L1)] 3+ 246 (22 720), 292 (5220), 363 (2700) [Gd(L2)] 3+ 292 (4020), 363 (1760) [Eu(L2)] 3+ 292 (3900), 363(1860) [Dy(L2)] 3+ 288 (7090), 363 (1840) [Yb(L2)] 3+ 279 (8780), 363 (918)

Figure 5 X

 5 Figure5X-Band EPR spectra of [Yb(L1)] 3+ in 0.5 mM water solution. Black lines, experimental spectra; red lines, simulation by using the parameters (S = 1), |D| = 300 MHz, giso = 2.00. Microwave Freq. 9.636 GHz; power, 0.2 mW, Mod. Amp. 0.4 mT, Freq. 100 KHz.

2

 2 This journal is © The Royal Society of Chemistry 20xx

Figure 6 X

 6 Figure 6 X-Band EPR spectra of (a) [Yb(L1)] 3+ and (b) [Dy(L1)] 3+ in 4 mM water solution after addition of 2 equiv. of ascorbate. Black lines, experimental spectra; red lines, simulation by using the parameters given in the text. Microwave Freq. 9.636 GHz; power, 2 mW, Mod. Amp. 1 mT, Freq. 100 KHz. T = 5 K.

Figure 7 .

 7 Figure 7. Relaxed PES scan of YL1 3+ and YL2 3+ (B3LYP/6-31g* (C,H,N,O) ECP (Y)).  is

Figure 8 .

 8 Figure 8. CEST spectrum of 14 mM solution of [Eu(L1)] 3+ under its nitroxide (black) and hydroxylamine (red) forms (reduced using sodium ascorbate). Recorded in a water:CD3CN (2:1) mixture at pH 7.4 (0.1 M HEPES buffer) at 11.7 T. T = 298 K, B1 = 25 μT, irradiation time of 3 s.

  ).

Figure 9 .

 9 Figure 9. FFC-NMR profiles of 2 mM solution of [Gd(L1)] 3+ under its nitroxide (black) and hydroxylamine (red) forms and [Gd(L2)] 3+ under its nitroxide (blue) and hydroxylamine (green) forms (reduced using sodium ascorbate). Recorded in water at pH 7.4 (0.1 M HEPES buffer) at 298 K.

  toxicity of complexes [Ln(L1)] 3+ and [Ln(L2)] 3+ has been investigated by MTT assays on M21 cell lines. The range of concentration tested was 1-40 M, with incubation times up to 72 h. The cell viability upon treatment with [Dy(L1)] 3+ and [Dy(L2)] 3+ is depicted in Fig. 9 (see ESI for the other compounds). Even after the longest incubation time the viability of the cells treated by up to 40 M [Ln(L1)]3+ was similar to that of the control sample without complex. Hence all the compounds of this series show negligible cytotoxicity, which is a pre-requisite for their use as imaging agents. Complexes of the [Ln(L2)]3+

Figure 9 .

 9 Figure 9. Cell viability after 48h treatment of M21 cells with increasing amounts of (left) [Dy(L1)] 3+ and (right) [Dy(L2)] 3+

  (Stelar s.r.l., Mede PV, Italy). The prepolarized (PP) and non-polarized (NP) sequences (see Fig. 1 of Ref. 49) were used below and above about 12 MHz, respectively. A high polarization field Bpol corresponding to a proton resonance frequency of 28 MHz was employed in the PP Please do not adjust margins

1-( 2 '

 2 ,2',6',6,'-tetramethyl-1'-oxyl-4'-piperidyl)-amide-1,4,7,10-Tetraazacyclododecane (1)

Table 1 .

 1 Electrochemical data of the ligands and complexes[a]

	Complex	E1/2 ox	Complex	E1/2

ox [Gd(L1)] 3+ 0.26 [Gd(L2)] 3+ 0.33 [Eu(L1)] 3+ 0.30 [Eu(L2)] 3+ 0.34 [Dy(L1)] 3+ 0.28 [Dy(L2)] 3+ 0.33 [Yb(L1)] 3+ 0.32 [Yb(L2)] 3+

				0.33
	L1	0.25	L2	0.31

  3+ does not show a CEST activity, which might be the consequence of different environment and mobility of the coordinated water molecule. The relaxivity of the gadolinium complexes is affected by change in oxidation state of the TEMPO arm, whereby r1 decreases by about 15% at 10 MHz upon reduction. It must be stressed that the influence of the nitroxide's redox state is about 10 times greater at low field than at high field.

	3. Conclusions
	In summary we have prepared two series of lanthanide(III)
	complexes (Gd, Eu, Dy, Yb) from two DOTA acetamide
	derivatives appended by one TEMPO arm. One features three
	acetamide groups ([Ln(L1)] 3+ ) while the other ([Ln(L2)] 3+ )
	harbours two acetamides and one alkyne group for further
	functionalization or vectorization through click chemistry. By
	luminescence measurements we demonstrate that both
	[Eu(L1)] 3+ and [Eu(L2)] 3+ possess one water molecule in their first
	coordination sphere, inferring that the metal ion is 9-or 8-
	coordinated, respectively. The distinct coordination polyhedron
	between the series is confirmed by EPR spectroscopy. The
	lanthanide complexes are redox-active in a physiological
	window, with the TEMPO being for instance readily reduced
	into hydroxylamine by sodium ascorbate. Complex [Eu(L1)] 3+ is
	CEST-active under its hydroxylamine form (CEST effect of 12 %).
	Under its nitroxide form the paramagnetism of the ligand
	accelerates spin relaxation, resulting in a significant quenching
	of the CEST effect. Interestingly, [Eu(L2)]

  H NMR (400 MHz, D2O) δ = 97.71, 94.42, 92.95, 19.51, 18.21, 16.86, 16.65, 15.97, 15.80, 15.24, 14.86, 12.31, 8.56, 4.12, 3.73, 1.57, 1.48, 0.13, -1.71, -24.16, -25.80, -26.42, -29.00, -31.27, -34.80, -53.HR-MS m/z calcd for C26H49O8N9F3GdS [M+CF3SO3] 2+ , This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins 431.130; found 431.131. [Dy(L1)] 3+ : from L1 (100 mg, 0.15 mmol) and dysprosium triflate (92 mg, 0.15 mmol). Yield: 27%. 1 H NMR (400 MHz, D2O) δ = 18.1, -2.6, -18.3; IR (cm -1 ): 3331, 3172, 2982, 1655, 1621, 1588, 1454, 1403, 1322, 1272, 1244, 1080, 632.

	92, -
	57.43, -58.00; IR (cm -1 ): 3361, 2981, 2927, 2867, 1667, 1626, 1458,
	1323, 1246, 1161, 1083, 1030, 924, 668, 639; MS-ESI m/z an
	appropriate isotope pattern was observed for C26H49F3O8N9SYb
	[M+CF3SO3] 2+ : 439.14; found, 440.77. [Eu(L1)] 3+ : from L1 (187 mg,
	0.27 mmol) and europium triflate (164 mg, 0.27 mmol). Yield: 49%.
	IR (cm -1 ): 3354, 3197, 2987, 1658, 1628, 1590, 1459, 1391, 1242,
	1225, 1173, 1079, 1027, 639. [Gd(L1)] 3+ : from L1 (30 mg, 0.06 mmol)
	and gadolinium triflate (34 mg, 0.06 mmol). Yield: 37%. IR (cm -1 ):
	3367, 3291, 3208, 2984, 2939, 2876, 1661, 1627, 1458, 1245, 1169,
	1027, 634;

2',2',6',6,'-tetramethyl-1'-oxyl-4'-piperidyl)-amide-4-propargyl- 1,4,7,10-Tetraazacyclododecane-1,7-bisacetamide (L2)

	H NMR (400 MHz, MeOD) δ = 3.45 (d, J = 2.2
	Hz, 1H), 3.38-3.36 (m, 2H), 3.30 (s, 2H), 3.19 (s, 2H), 2.76-2.56 (m,
	16H); 13 C NMR (100 MHz, MeOD) δ = 176.4, 168.2, 77.9, 75.6, 59.2,
	53.4, 53.0, 51.3, 46.8, 39.7, 30.7; IR: 3283, 2962, 2842, 2689, 2464,
	2062, 1654, 1586, 1457, 1372, 1341, 1118, 973, 758; HRMS (m/z):
	[M+H] + calcd for C15H29N6O2, 325.235; found, 325.235.
	1-(

  -1 ): 3369, 3244, 2958, 1667, 1606, 1454, 1233, 1172, 1029, 647; ESI-MS m/z an appropriate isotope pattern was observed for [M] 3+ : C26H47N8O4Eu, 229.43; found, 229.39. Elemental analysis: calculated for C26H47N8O4EuI2Cl.4H2O; C: 29.76, H: 5.30 N: 10.68, observed C: 29.71 H: 5.30 N: 10.62. [Yb(L2)] 3+ : from L2 (300 mg, 0.45 mmol) and ytterbium triflate (281 mg, 0.45 mmol). Yield: 96%. 1 H NMR (500 MHz, MeOD) δ = 77.8, 65.8, 57.9, 46.5, 36.9, 22.4, -5.3, -15.7, -17.4, -32.5, -53.2, -83.1; IR (cm -1 ): 3376, 3270, 2975, 2945, 2876, 1670, 1627, 1463, 1243, 1150, 1081, 1028; ESI-MS m/z : from L2 (300 mg, 0.45 mmol) and gadolinium triflate (274 mg, 0.45 mmol). Yield: 84%. IR (cm -1 ): 3379, 3257, 2972, 2868, 1664, 1615, 1455, 1167, 1080, 1019, 632. [Dy(L2)] 3+ : from L2 (300 mg, 0.45 mmol) and gadolinium triflate (276 mg, 0.45 mmol). Yield: 87%. IR (cm -1 ): 3364, 3274, 2969, 2874, 1667, 1618, 1459, 1246, 1174, 1079, 1024, 635.

	an	appropriate	isotope	pattern	was	observed	for
	C26H47N8O4Yb(CF3SO3)4, [M+4 CF3SO3] -: 1305.12; found, 1304.89.
	[Gd(L2)]

3+

Acknowledgements

The authors thank the French National Research Agency in the framework of the "Investissements d'avenir" program (ANR-15-IDEX-02), Labex ARCANE and CBH-EUR-GS (ANR-17-EURE-0003) and the French National Agency for Research, program Co-Lantha (ANR-17-CE07-0034) for financial supports. This work was performed under the auspices of the COST Action AC15209, EURELAX. The authors are grateful to Dr. Pierre Girard for technical assistance for the DFT calculations and the Centre de Calcul Intensif en Chimie de Grenoble (CECIC) for providing the computational resources. The ICMG Platform (FR 2607) is acknowledged for the analytical support.

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 11

Please do not adjust margins Please do not adjust margins 4.6 MTT assay M21 cells were seeded into 96-well plates (1.5 × 10 3 cells per well) in 100 μl of culture medium. After 24 h, cells were treated with the lanthanide(III) complexes at various concentrations. Following incubation for 24 h, 48 h or 72 h, 10 μl of a MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) stock solution (Euromedex, Mundolsheim, France) in PBS at 5 mg ml -1 was added in each well and the plates were incubated at 37 °C for 2 h. To solubilize water-insoluble purple formazan crystals, SDS 10% / HCl 0.1% solution was used. After 24 h, absorbance was measured on an ELISA reader (Tecan, Männedorf, Switzerland) at a test wavelength of 570 nm and a reference wavelength of 650 nm.

EPR spectroscopy

M21 cells were seeded in 4-well plates (1 x 10 5 cells per well) in 1 mL of culture medium. After 24 h, cells were treated with the complexes at 200 μM concentration during 5 min, 10 min, 30 min or 1 h. Cell culture medium was recovered and M21 cells where harvested. A fraction of M21 pellets was then washed twice in cold PBS and incubated in lysis buffer (10mmol/L Tris-HCl pH 7.5, 120 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, 0.5% Nonidet P-40, 0.05% sodium dodecyl sulfate, supplemented with protease inhibitors (Na3VO4 and NaF). After 10 min on ice lysates were centrifugated at 20,000 x g for 15 minutes, and soluble fraction was recovered. Cell culture medium, pellets of intact M21 cells or M21 cell lysates were then analyzed by EPR spectroscopy.

Conflicts of interest

There are no conflicts to declare.


Anthony Rodrigues

Jean-Michel Tatibouët

Elodie Fourre
email: elodie.fourre@univ-poitiers.fr

Operando DRIFT spectroscopy characterization of intermediate species on catalysts surface in VOC removal from air by non-thermal plasma assisted catalysis

Keywords: In-situ characterization, infrared spectroscopy, non-thermal plasma, VOC

An innovative plasma discharge reactor was developed to fit an infrared cell and to allow the in situ characterization of isopropanol (IPA) and toluene decomposition at the surface of three metal oxides (-Al2O3, TiO2 and CeO2). The impact of the plasma discharge on the conversion of these pollutants, with the material placed in the discharge area, was studied under real time conditions at atmospheric pressure via infrared analysis. The plasma treatment of IPA molecules led to the formation of acetone, propene, acetic acid and/or formic acid. By contrast, the toluene oxidation led to the rapid opening of the aromatic ring, followed by the total oxidation through carboxylic formation of the species arising from the toluene molecules fragmentation.

Introduction

Non thermal atmospheric plasma (NTAP) is now recognized as an innovative technology applied to a wide range of applications (etching, deposition of thin film, volatile organic compound oxidation, surface functionalization, biomedical...) [1]. The advantageous properties of NTAP, in terms of energetic and environmental aspects, are responsible for the enhanced research in this field. Non thermal atmospheric plasmas are produced by a variety of electrical discharges or electron beams leading to plasmas such as corona discharges, gliding arc discharges, dielectric barrier discharges (DBD), plasma needle, plasma jets and micro hollow cathode discharges [2]. Non thermal plasmas are generated by the application of a high potential difference between two electrodes that leads to a strong electrical field, resulting in the formation of a highly reactive environment favorable to various reactions. For decades, non-thermal atmospheric plasma was used mainly for the production of ozone and water purification [3]. More recently, due to a greater concern on environmental issues, research on waste gas treatment, volatile organic compounds (VOCs) removal has been largely studied. In particular, the presence of a catalyst in the plasma discharge resulted in a synergistic effect and improved VOCs conversion [4][5][6]. However, if research has shown tremendous results on VOCs conversion, a few points remain unclear. It concerns essentially the interactions between the chemical species generated in the plasma discharge and a material surface. The willingness to identify active species in the plasma discharge is very challenging. It is a complex field of research, as the species concentration, nature and energetic power are dependent of the plasma reactor configuration, chemical and electrical parameters. Already, research has been dedicated to in situ identification of plasma species in the gaseous phase by spectroscopic methods [7][8][9][10][11][12][13][14][15]. Most of these studies are dealing with the characterization of the plasma species but not with their reactivity. Rivallan et al. [11] have studied the evolution of air/CO2 gas mixture under non-thermal plasma by in situ FTIR at the microsecond time-scale. They showed that the mechanism of CO2 consumption is reversible and that CO2 molecules are excited through a collision with excited N2 molecules, where the transfer of energy occurs by a resonant effect. Other authors tried to elucidate some aspects of the ion chemistry occurring in dielectric barrier and corona discharges with benzene/air [7] or different gases such as gas mixtures including Ar, H2, N2, O2 and CH4 as plasma precursors [8]. OH radical concentration was determined by indirect detection with salicylic acid [13] and direct measurement by laser-induced fluorescence [12]. Kim et al. [15] have reviewed recent progresses in understanding the interaction of NTP and catalysts. They insisted on the effects of the electrical characteristics of the catalyst on the plasma discharge generation and therefore chemical reactivity. In a recent publication [14], we have shown that the decomposition of isopropanol (IPA) by non-thermal plasma on -Al2O3, analyzed by operando Fourier Transform Infrared (FTIR) under low vacuum could proceed via oxidation, aldolization and fragmentation mechanisms. Such cascade reactions were only possible when -Al2O3 catalyst was placed in the discharge zone. This VOC has been studied and published in the literature with results essentially based on the analysis of the outlet gas composition. IPA conversion leads to the formation of acetone, acetic acid, formaldehyde (when the reaction is incomplete), formic acid, CO2 and H2O [14][15][16][17][18][19]. Christensen et al. [20] reported a spectroscopic study of non-thermal plasma in an air-fed dielectric barrier discharge plasma jet. In situ analysis of the plasma glow and downstream gas by FTIR revealed the presence of various species such as O3, N2O5, N2O, HNO3, CO2, CO and a vibrationally excited form of CO2 (i.e. CO*2 (v)).

Here we are dealing with a non-thermal plasma reactor set at atmospheric pressure and coupled to reflectance infrared spectroscopy. The test reactions studied in this work concerned the removal of isopropanol and toluene from -Al2O3, TiO2 and CeO2 surfaces by non-thermal plasma. The impact of the plasma discharge on the conversion of these pollutants, with the material placed in the discharge area, was studied under real time conditions at atmospheric pressure via infrared analysis.

Experimental

A non-thermal plasma at atmospheric pressure (NTPAP) reactor was developed and optimized for its combination with a DRIFT spectrometer. The spectrometer was modified in order to carry out a DBD plasma in the DRIFT cell while a direct surface analysis was carried out. The cell was made of Polytetrafluoroethylene (PTFE) and included a cylindrical base (30 mm diameter, 29 mm height) and a conical top (26 mm height). The cone was equipped with two ZnSe windows, to allow the passage of the IR beam, and one glass window (figure 1a) to view the discharge. The volumic dielectric barrier discharge plasma consisted in a tip electrode (figure 1b, tip diameter 0.5 mm) held in the centre of the cone and a counter electrode (10 mm  10 mm copper tape) placed under a dielectric (0.5 mm thick in PEEK: polyether ether ketone). The sample was placed on the dielectric, underneath the tip. The gap between the tip and dielectric surface was kept constant (1 mm). The electrodes were connected, via a capacitor of known value (Cm= 0.56 nF), to a high voltage power amplifier (Trek, 20/20A) coupled to a function generator (TTi TG1010A). Experiments were carried out in an airtight cell with the possibility to vary the chemical and electrical parameters. FTIR Spectrometer Frontier (PerkinElmer, UK), equipped with a Mercury Cadmium Telluride (MCT) detector, was used to record IR spectra in the 4000-900 cm -1 range with a resolution of 8 cm -1 . Each spectrum is the accumulation of 50 analyses allowing an acquisition time below 1 min. The contributions of gaseous H2O and CO2 were subtracted when scanning. Before each experiment the cell was purged with dry air for 10 min (30 mL.min -1 ) before the initiation of the plasma discharge. IR spectra of the sample wafer were then recorded in reflection mode (DRIFT: Diffuse Reflectance Infrared Fourier Transform) as a function of the duration of DBD plasma. Scans were taken while the plasma treatment was switched on and over a period of time going from 1 min to 8 hours. All the experiments were carried out at atmospheric pressure.

The DBD experiments were studied under "dynamic" conditions, a flow rate of 30 mL.min -1 of gas (dry air, Air liquide) was constantly running in the cell chamber.

The power injected in the reactor was determined by the analysis of the Lissajous figure (figure 2) that reports the transferred electrical charge Qm as a function of the applied voltage (Ua) using the so-called "Manley method" [21]. Qm was calculated from the measured voltage (Um) across the capacitor (Cm) connected in series to the ground electrode. The energy injected, E (per cycle) was calculated from the integration of the area formed by the Lissajous figure. The energy allowed the calculation of the power (in W) injected in the reactor as well as the specific energy Espe (in J.L -1 ) following equation 1 and 2, where f is the frequency (in Hz) and D the gas flow rate in (L.s -1 ):

𝑃 = 𝐸 × 𝑓 (1)
(2)

D P E spe 
The experiments were carried out at 16 kV (500 Hz), where the plasma discharge was stable, homogeneous and sufficiently energetic to promote excited species. These parameters correspond to a power of 24 mW and an energy density of 48 J.L -1 .

The catalyst sample was in the form of a thin layer of powder (50-100 mg) slightly pressed on the dielectric plate in order to avoid its spraying under the effect of gas flow rate and plasma. The three oxides were commercial oxides (table 1). The specific surface area of the samples was determined by the BET (Brunauer-Emmett-Teller) method from the nitrogen adsorption isotherms at -196 C in an automated Micromeritics Tristar 3000 apparatus after drying for 8 hours at 400 C.

Results

Electrical parameters of the plasma generated in the IR cell were determined with and without the presence of a catalytic material. The influence of the material on the plasma discharge was evaluated by comparing the variation of the plasma power as a function of applied voltage (Figure 3). A slight decrease of the power is noticed when Al2O3 is placed in the discharge, likely due to the slight modification of the dielectric properties of the dielectric plate when covered by alumina.

Isopropanol removal

All the surfaces were first placed in contact with a gas mixture air/IPA (100 ppm) at atmospheric pressure for 15 minutes. This was followed by a 10 min flow of air prior to the plasma treatment, also under air flow. Figure 4A displays the evolution of the IR spectra of Al2O3 as a function of treatment time, from 1 to 60 minutes. The same experiment was carried out on the two other oxide surfaces of CeO2 and TiO2 (figures 5A and 6A). Additionally, spectra of three likely products (acetic acid, formic acid and acetone) were recorded and are displayed in figures 4B, 5B and 6B. The spectra were recorded after 10 mins adsorption of a mixture of air/ product (100ppm) at atmospheric pressure followed by a 10 mins purge under air flow.

The IR spectra were obtained after subtraction of the background spectrum of the sample under air before adsorption. For the three samples, after IPA adsorption and before plasma treatment, the predominant bands corresponding to the C-H asymmetric and symmetric stretching (-as and -s) were detected between 3030 and 2840 cm -1 and between 1510 and 1300 cm -1 for the methyl C-H asymmetric and symmetric bends (-as and -s). Between 1255 and 1205 cm -1 , -O-H from un-dissociated IPA was identified. Other components, with lower intensity, around 1150 and 1075 cm -1 were related to the C-O stretch and C-C skeletal of isopropoxide species, formed via dissociative adsorption of the IPA molecule or/and to molecularly adsorbed IPA [16,[22][23][24][START_REF] Coates | Interpretation of infrared spectra, A practical approach[END_REF]. The negative bands in the 3750-3600 cm -1 region, corresponding to the surface hydroxyl groups of metallic oxides show that IPA adsorption can occur either as isopropoxy group (scheme 1-a) or by coordination with surface OH groups (scheme 1-b).

The remaining negative band even after oxidation of IPA by non-thermal plasma strongly suggests that the IPA is mostly adsorbed as an isopropoxy species (scheme 1-b) rather than by coordination with surface OH groups [START_REF] Al-Abadleh | [END_REF][27][28]. As soon as the discharge was initiated, the bands corresponding to the methyl group stretching (2975-2840 cm -1 region) decreased as a function of the plasma treatment time, whatever the sample. After 60 minutes of plasma treatment, a residual C-H contribution is still visible which results from the formation of decomposition compounds such as acetone, aldehydes and/or acids. A very broad band going from 3340 to 2250 cm -1 emerged after 5 minutes of treatment for TiO2 and CeO2 and 20 minutes for -Al2O3 which corresponds to the O-H stretch of carboxylic acid. This band is prominent on -Al2O3 and similarly, but less evident, on TiO2 and CeO2.

In the lower wavenumber region, the analysis is more difficult due to the overlapping of the bands. However, the progressive formation (from 1 to 60 min treatment) of a band corresponding to a C=O group (ketone, aldehyde and/or carboxylic acid) and centered at 1730, 1760 and 1712 cm -1 for -Al2O3, TiO2 and CeO2, respectively, is clearly identified. Regarding -Al2O3 and TiO2, a shoulder within the ketone region band is visible at a lower wavenumber (1695 and 1730 cm -1 ) and correlates closely to the C=O band of acetic and/or formic acids.

CeO2 presents a much wider band, centered at 1712 cm -1 , encompassing the C=O contribution of the acids.

Bands of methyl bending vibrations (δ) evolved differently. On -Al2O3 surface, an increase and a broadening of the -as C-H band (1465 cm -1 ), a decrease/shift of -s C-H band (IPA: 1382 cm -1 ) to 1360 cm -1 were observed as a function of the treatment time. The band at 1360 cm - 1 , emerging after 5 minutes, is related to the methyl bending vibrations of acetone [29][30][31].

The bands at 1595 and 1465 cm -1 could be assigned to the C=C stretching and C-H bending mode of methyl or methylene group of propene, respectively [START_REF] Benaliouche | Micropor Mesopor Mat[END_REF], according to the products formation observed by IPA oxidation at low temperature on pure -Al2O3 [START_REF] Lakshmanan | [END_REF]. Simultaneously, a small shoulder at 1225 cm -1 also appeared after 20 minutes treatment and the large band at 1730 cm -1 was assigned to acetone formation. The small residual contribution remaining at 1150 cm -1 could correspond to C-O bonds stretching and assign to formic acid formation. These observations show that isopropoxide species formed by the dissociative adsorption of isopropanol give rise to acetone and formic acid on -Al2O3 when treated by non-thermal plasma under air.

On TiO2, an increase and a broadening of the -as C-H band (1470 cm -1 ) and the total disappearance of -s C-H band (1385 cm -1 ), were observed as a function of the plasma treatment time. The IPA bands at lower wavenumbers: -O-H (1253 cm -1 ), C-O stretching and C-C skeletal elongation (1150 and 1075 cm -1 ), progressively weakened till complete disappearance as the plasma treatment time increased showing that IPA on TiO2 is rapidly oxidized into acetone followed by further decomposition into CO2 and gas phase water. A small acidic residue is visible from the broad band in the 3000 cm -1 region. Additionally, at 1470 cm -1 and in smaller extend at 1590 cm -1 bands were identified and corresponded to the C-H bending and the COO -stretching, respectively. On CeO2, the bands corresponding to the -as (1470 cm -1 ) and -s (1390 cm -1 ) bending mode of methyl are evolving and broadening as a function of the plasma treatment time. In the same time, a large band is growing at 1275 cm -1 which corresponds to the O-H bending vibration mode of carboxylic acids. As for the two other samples, it is difficult to differentiate the different acids and their contribution due to the overlapping of the bands in this region.

Cerium oxide that shows particularly broad bands makes it difficult to observe the evolution of acetone formation/oxidation on this surface. Finally, an interesting feature, only visible on CeO2 is the adsorption/desorption of CO2 in the 2300-2400 cm -1 region (2365 and 2333 cm -1 ), that may arise from the decomposition of IPA into CO2. However, we have to be careful with this interpretation since published work on CO2 adsorption on -Al2O3 [34] or tin oxide [35] revealed the presence of bands in the 1900-1200 cm -1 range relative to adsorbed CO2, which may, in this case, be hidden by other contributions at low wavenumbers.

According to these observations, the steps following IPA stabilization on the surface can follow these two pathways: either its oxidation into acetone, either a C-C bond breakage of a methyl group. Oxidation reactions taken place here can either come from a radical attack, probably from O  or  OH, on the -OH group of IPA, either via redox reactions of the adsorbed isopropoxide species on the metal oxide surface acid sites. C-C bond breakage can be achieved by  OH radical attack and the resulting methyl radical can react with oxygenated species to form formaldehyde, then formic acid. The other radical fragment, CH3CH  OH, can then be oxidized in acetaldehyde, then in acetic acid. On alumina, the formation of propene from IPA dehydration can also occurs, followed by its decomposition into CO2 and H2O.

Toluene removal

The experiments regarding toluene elimination were performed in the same conditions as for IPA removal. Toluene was previously adsorbed on the catalysts (100 ppm in air for 10 min, 30 mL.min -1 ) followed by purging with dry air before the start of the plasma treatment. The recorded spectra as a function of time are presented in figure 7 (A to C). As expected, on the three oxides the main reference IR bands of adsorbed toluene are visible at around 3100-3000 cm -1 domain corresponding to the C-H aromatic (3030 and 3080 cm -1 ) and at 2930 and 2880 cm -1 corresponding to the C-H stretching of the methyl group and at 1495, 1460 and 1610 cm - 1 , corresponding to the C-C stretching in the aromatic ring. The negative bands at 1705 (bending mode of hydrogen bonded surface hydroxyls) and 3695 cm -1 (stretching mode) could be assigned to the loss of surface hydroxyl group upon toluene adsorption [36][37][38][39][40]. On CeO2 and to a lesser extent on -Al2O3, a small contribution of in plane C-H bending at 1081 and 1035 cm -1 was identified as well as weak bands at 1160 and 1180 cm -1 for CeO2 and -Al2O3, respectively. Finally, a weak overtone in the 2000-1800 cm -1 region is detected.

As soon as the plasma was generated, and this independently of the catalyst sample tested, the C-H aromatic bands (3030 and 3080 cm -1 ) decreased rapidly after only one minute of plasma treatment and this until 10 minutes. At 10 minutes, no C-H aromatic contribution is visible, and this for the three surfaces, indicating the complete transformation of toluene.

In the same time, a strong band in the 1730-1755 cm -1 region evolved after 5 minutes of plasma treatment characteristic of the presence of a C=O bond. As the plasma treatment time increased, the band increased, shifted to higher wavenumber after 10 minutes of plasma to reach 1775, 1770 and 1790 cm -1 on -Al2O3, CeO2 and TiO2, respectively (table 2 and figure 7).

The non-symmetry of the bands and apparition of a shoulder (1700-1725 cm -1 ) at 10 minutes for CeO2 and TiO2 and 20 minutes for -Al2O3 indicate the presence of a carboxylic acid in addition to aldehyde or ester [START_REF] Coates | Interpretation of infrared spectra, A practical approach[END_REF][START_REF] Larkin | IR and Raman Spectroscopy: Principles and Spectral Interpretation[END_REF]. The bands at 1330, 1250 and 1290 cm -1 on -Al2O3, TiO2 and CeO2, respectively, can be attributed to C-O stretching. In addition, the broad shoulder of O-H stretching (2500-3500 cm -1 ) confirms the presence of carboxylic acid. Independently of the catalyst, the growing of the 1790-1770 cm -1 band is associated with the formation of a negative signal in the OH vibration domain (3500-3700 cm -1 ) showing that the species formed under plasma adsorb on OH surface groups. By increasing the plasma treatment duration, the main change is the shift of the C=O band for more than 10 minutes of plasma. This shift could be assigned to the formation of bridged carbonates [42] on the catalysts surface showing the total oxidation of toluene under plasma treatment.

A band at 1345 cm -1 appeared after one minute and could also correspond to primary O-H bending (-OH) or =C-OH phenol stretching. The band increased and shifted to 1330 cm -1 until 20 minutes and remained constant until 60 minutes. At this wavenumber, the functional group identified is more likely C-O from carboxylic acid or =C-OH phenol stretching but also N-O symmetrical stretching from olefinic compounds. However, other nitrogen related contribution were not identified. On CeO2, less intense bands appear at 1414 and 1250-1216 cm -1 which could be due to the formation of carboxylic acids (O-H bending and C-O stretching, respectively) and at 1610 cm -1 which could be assigned to the formation of adsorbed formate species [START_REF] Köck | [END_REF]. The relative intensity of this band decreases from -Al2O3, TiO2 to CeO2, following the increasing oxidative property (or basicity) of these oxides.

On the three surfaces, the band of C=C stretching at 1460 cm -1 and at 1610 cm -1 broadened and increased until 60 minutes while the one at 1495 cm -1 decreased until complete disappearance. The increasing and broadening of the 1460 cm -1 band could be due to various stretching behavior such as C=C of phenol or C=C from alkenes indicating a rupture of the aromatic ring. A weak shoulder in the 2950-2930 cm -1 region, indicates the presence of C-H stretching from methyl group only, without aromatic contribution. The presence of bands in the 2300-2400 cm -1 domain is associated to the presence of adsorbed CO2.

According to the results of various authors [44][45][46][47][48][49][50][51][52] and ours, we can propose some probable reaction mechanisms for toluene elimination. First of all, Kohno et al. [44] showed that, from the 3 pathways undertaken by NTAP species for VOC elimination, the electron impact would be the favored one as it presents the lowest reaction rate constant in the order of 10 -6 cm 3 .s - 1 . The two other pathways, ion collisions and radical attack (O  /OH  ) would occur subsequently and contribute to toluene oxidation and ring opening reactions. The electronic impact would lead to the formation of benzene radical that would rapidly be oxidized in phenol. For the later, the aromatic cycle would be rapidly hydroxylated into hydroquinone, further oxidized in benzoquinone. Finally the ring would be opened by oxygen or OH radicals leading to the formation of aldehydes and carboxylic acids with different carbon chains length. Possible mechanisms are displayed in figure 8. As long as oxidative radicals are present, in addition to electronic impact, the reaction would continue until complete fragmentation into CO2 and H2O following various mechanisms that are currently not completely identified and still need further investigation. Additionally, methyl radical would be oxidized in formic acid followed by complete elimination in CO2/H2O. It seems that the oxidation of the methyl group on toluene, leading to benzaldehyde and benzoic acid, is not the favored pathway undertaken for toluene oxidation by non-thermal plasma. In fact, from our results, it seems that the band related to C=O apparition coincides with the complete disappearance of aromatic C-H bands, indicating a rapid opening of the aromatic ring prior to the oxidation of the fragments into carboxylic acid.

Conclusions

This new infrared cell allows characterizing by DRIFT the adsorbed organic species on a catalyst surface under DBD non-thermal plasma and in controlled gaseous environment at atmospheric pressure, realizing then a true operando analysis of a catalyst surface submitted to the action of a non-thermal plasma. The method has proven to be so efficient that it was possible to follow the evolution of secondary compounds, arising from the oxidation of isopropanol and toluene. The plasma treatment of IPA molecules adsorbed on -Al2O3, TiO2 and CeO2 surfaces led to the formation of acetone, propene, acetic acid and/or formic acid. On CeO2 and TiO2 surface, only acids remained after 60 minutes of plasma treatment while -Al2O3 showed a small presence of acetone and propene on its surface. By contrast, the toluene oxidation by non-thermal plasma led to the rapid opening of the aromatic ring, followed by the total oxidation through carboxylic formation of the species arising from the toluene molecules fragmentation. These results should be considered as preliminary experiments probing the extended possibilities of this new IR cell to characterize in operando conditions a catalyst or any surface submitted to a non-thermal plasma and are able to improve the knowledge on plasma material interactions.

Figures captions Scheme 1 :Figure 1 (

 11 Figures captions

Figure 1 (

 1 Figure 1 (B): Views of the disassembled cell (a to d).

Figure 2 :Figure 3 :

 23 Figure 2: Lissajous curve at Ua = 16kV and f = 500 Hz

Figure 4 :Figure 5 :Figure 6 :Figure 7 :

 4567 Figure 4: DRIFT Spectra on -Al2O3 (A) In situ IR spectra of the evolution of adsorbed isopropanol as a function of plasma treatment time from bottom to top (adsorbed IPA then 1, 5, 10, 20, 30, 45 and 60 min of plasma treatment) (B) Spectra of adsorbed IPA after 60 min of plasma treatment and adsorbed acetone, acetic acid and formic acid (from bottom to top)

Figure 8 :Figure 1 (

 81 Figure 8: Possible mechanism pathways and intermediates of toluene oxidation and degradation

Figure 1 (Figure 2 :Figure 3 :

 123 Figure 1 (B): Views of the disassembled cell (a to d).

Figure 4 : 20 Figure 5 : 21 Figure 6 :

 4205216 Figure 4: DRIFT Spectra on -Al2O3 (A) In situ IR spectra of the evolution of adsorbed isopropanol as a function of plasma treatment time from bottom to top (adsorbed IPA then 1, 5, 10, 20, 30, 45 and 60 mins of plasma treatment) (B) Spectra of adsorbed IPA after 60 mins of plasma treatment and adsorbed acetone, acetic acid and formic acid

Figure 7 :

 7 Figure 7: In situ DRIFT spectra of the evolution of toluene on A) -Al2O3, B) CeO2 and C) TiO2 as a function of plasma treatment time from bottom to top (adsorbed toluene then 1, 5, 10, 20, 30, 45 and 60 min of plasma treatment).

Figure 8 :

 8 Figure 8: Possible mechanism pathways and intermediates of toluene oxidation and degradation

Table 1 :

 1 Catalysts characteristics

	Oxide	Supplier	Surface area (m 2 .g -1 )
	-Al2O3	Degussa	102
	TiO2 P25	Sigma Aldrich	9
	CeO2	Prolabo	23

Table 2 :

 2 Shift of the C=O band as a function of plasma treatment time and corresponding chemical groups[START_REF] Coates | Interpretation of infrared spectra, A practical approach[END_REF]34].

	Plasma time	0 1	5	10	20	30	45	60
	(min)
		Wavenumber maxima (cm -1 )
	Al2O3 -1740	1740	1745	1769	1775 1775 1775
	Corresponding	Aldehyde/	Ald/ester Ald/ester Ald/ester/	Carb.
	carbonyl	ester			acid
	group
	CeO2 -1736	1736	1760	1783	1770 1770 1770
	Corresponding	Aldehyde/	Ald/	Ald/ester/	Carb.
	carbonyl	ester	ester	acid
	group
	TiO2 -1750	1750	1775	1782	1790 1790 1790
	Corresponding	-Aldehyde/	Ald/	Ald/ester/	Carb.
	carbonyl	ester	ester	acid
	group

Ald: aldehyde; carb: carbonate

Acknowledgments

The authors would like to thank the French Ministry of Research for the funding of the PhD grant of A. Rodrigues.