Clarisse Quignon
email: c.quignon@inci-cnrs.unistra.fr

Matthew Beymer

Karine Gauthier
email: karine.gauthier@ens-lyon.fr

Francois Gauer
email: gauer@inci-cnrs.unistra.fr

Valerie Simonneaux
email: simonneaux@inci-cnrs.unistra.fr

François Gauer

Valérie Simonneaux

Valérie Simonneaux

François Gauer

Thyroid hormone receptors are required for the melatonin-dependent control of Rfrp gene

Keywords: seasonal rhythms, deiodinase, tanycytes, neuropeptides, TSH Vimentin TRα Merge

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INTRODUCTION

Most species adjust biological and behavioral functions to align with the seasonal variations of their environment. This adaptive process is particularly critical to ensure that birth and weaning of the offspring occur around springtime when environmental conditions are optimal for the survival. Early studies, mostly performed on seasonal breeders such as Syrian or Siberian hamsters, and sheep, have highlighted the pivotal role of the hormone melatonin in synchronizing physiology with the seasons [START_REF] Reiter | Photoperiod: its importance as an impeller of pineal and seasonal reproductive rhythms[END_REF][START_REF] Rollag | Pineal melatonin content in male hamsters throughout the seasonal reproductive cycle[END_REF]. Indeed, under the control of the master hypothalamic clock, melatonin synthesis occurs at night only so that in long winter nights (or short photoperiod, SP) melatonin production is longer than in short summer nights (or long photoperiod, LP). Numerous experiments of pineal lesions or exogenous melatonin administration have formally demonstrated that it is the duration of nocturnal melatonin release which triggers the physiological response to seasonal change [START_REF] Hazlerigg | Seasonal reproduction in mammals[END_REF].

In the last fifteen years, major progresses has been made in determining how this melatonin signal is integrated [START_REF] Hazlerigg | Seasonal reproduction in mammals[END_REF][START_REF] Dardente | An integrative view of mammalian seasonal neuroendocrinology[END_REF]. The long SP melatonin peak inhibits expression of the β subunit of thyroid stimulating hormone gene (Tshb) in the pituitary pars tuberalis so that the melatonin signal is decoded into photoperiodic changes in TSH production with higher levels in LP compared to SP [START_REF] Bockmann | Short photoperiod-dependent down-regulation of thyrotropin-alpha and -beta in hamster pars tuberalis-specific cells is prevented by pinealectomy[END_REF][START_REF] Dardente | MT1 melatonin receptor mRNA expressing cells in the pars tuberalis of the European hamster: effect of photoperiod[END_REF]. Pars tuberalis TSH in turn binds to receptors located on the tanycytes, specific glial cells lining the basal part of the third ventricle, triggering an increase in deiodinase 2 (Dio2) and a decrease in deiodinase 3 (Dio3) gene expression [START_REF] Yoshimura | Lightinduced hormone conversion of T4 to T3 regulates photoperiodic response of gonads in birds[END_REF][START_REF] Watanabe | Photoperiodic regulation of type 2 deiodinase gene in Djungarian hamster: possible homologies between avian and mammalian photoperiodic regulation of reproduction[END_REF][START_REF] Revel | Melatonin regulates type 2 deiodinase gene expression in the Syrian hamster[END_REF][START_REF] Barrett | Hypothalamic thyroid hormone catabolism acts as a gatekeeper for the seasonal control of body weight and reproduction[END_REF][START_REF] Ono | Involvement of thyrotropin in photoperiodic signal transduction in mice[END_REF][START_REF] Nakao | Thyrotrophin in the pars tuberalis triggers photoperiodic response[END_REF][START_REF] Hanon | Ancestral TSH mechanism signals summer in a photoperiodic mammal[END_REF][START_REF] Klosen | TSH restores a summer phenotype in photoinhibited mammals via the RF-amides RFRP3 and kisspeptin[END_REF]. As Dio2 converts thyroxine (T4) to the bioactive triiodothyronine (T3) and Dio3 inactivates T3, the melatonin-driven TSH-induced switch in Dio2/Dio3 leads to higher level of intrahypothalamic T3 in LP as compared to SP. This photoperiodic change in the TSH/T3 signal is pivotal for seasonal synchronization of reproduction as along with body weight and temperature [START_REF] Barrett | Hypothalamic thyroid hormone catabolism acts as a gatekeeper for the seasonal control of body weight and reproduction[END_REF][START_REF] Klosen | TSH restores a summer phenotype in photoinhibited mammals via the RF-amides RFRP3 and kisspeptin[END_REF][START_REF] Murphy | Effects of manipulating hypothalamic triiodothyronine concentrations on seasonal body weight and torpor cycles in Siberian hamsters[END_REF][START_REF] Bank | Alternation between short-and long photoperiod reveals hypothalamic gene regulation linked to seasonal body weight changes in Djungarian hamsters (Phodopus sungorus)[END_REF]. However, until now the cellular targets through which T3 synchronizes reproductive and metabolic functions have not yet been established.

Our previous studies have demonstrated the pivotal role of two neuropeptides known to regulate the activity of the GnRH neurons: kisspeptin (Kp), expressed in neurons of the arcuate nuclei, and argininephenylalanine-amide-related peptide 3 (RFRP-3), expressed by neurons in the dorsomedial hypothalamus (DMH) [START_REF] Simonneaux | A Kiss to drive rhythms in reproduction[END_REF][START_REF] Angelopoulou | Functional Implications of RFRP-3 in the Central Control of Daily and Seasonal Rhythms in Reproduction[END_REF]. Both neuropeptides exhibit robust photoperiodic variations, with a highly conserved SPinhibition of RFRP-3 expression across seasonal species. Chronic central infusion of Kp [START_REF] Revel | Kisspeptin mediates the photoperiodic control of reproduction in hamsters[END_REF][START_REF] Cázarez-Márquez | Kisspeptin and RFRP3 modulate body mass in Phodopus sungorus via two different neuroendocrine pathways[END_REF] or RFRP-3 [START_REF] Ancel | Stimulatory effect of RFRP-3 on the gonadotrophic axis in the male Syrian hamster: the exception proves the rule[END_REF][START_REF] Henningsen | Roles of RFRP-3 in the Daily and Seasonal Regulation of Reproductive Activity in Female Syrian Hamsters[END_REF] is able to restore gonadal activity of hamsters kept in inhibitory SP conditions. Furthermore, chronic central administration of TSH in SP-adapted hamsters not only restores the LP-phenotype of gonadal activity but also that of Kp and RFRP-3 expression [START_REF] Klosen | TSH restores a summer phenotype in photoinhibited mammals via the RF-amides RFRP3 and kisspeptin[END_REF]. From these observations, we hypothesized that the melatonin-controlled hypothalamic T3 could act directly on Kp or RFRP-3 neurons to regulate seasonal reproduction.

The lack of genetically modifiable seasonal models such as hamsters or sheep has strongly hampered the investigation into the cellular and molecular mechanisms of seasonality. Unfortunately, the classic mouse models using C57BL/6J mice are melatonin-deficient, and melatonin proficient strains of mice such as CBA/N and C3H do not express overt seasonal phenotype. C57BL/6J mice however express melatonin receptors and acute melatonin administration has been shown to regulate Tshb and Dio2/3 expression [START_REF] Ono | Involvement of thyrotropin in photoperiodic signal transduction in mice[END_REF].

In this study, we hypothesized that genetically modified C57BL/6J mice may help us better understand the molecular mechanism underlying melatonin action on the hypothalamic circuits regulating reproduction. We first showed that expression of Tshb and Dio2/3 as well as Rfrp, the gene encoding RFRP-3, are regulated by exogenous administration of melatonin with a nighttime (SP-like) in C57BL/6J mice, in a similar way as in CBA mice exposed to SP. Further, we demonstrated that a global, but not a neuron-specific, mutation of the thyroid-hormone receptor alpha (TRα) in C57BL/6J mice, selectively abolishes the melatonin regulation of Rfrp expression, but not that of Tshb and Dio2/Dio3. Finally, we found that TRα is widely co-expressed with vimentin in the tanycytes. Altogether our studies indicate that non-neuronal T3 signaling, possibly in the TRα-expressing tanycytes, is required for the melatonin regulation of Rfrp expressing neurons.

MATERIAL AND METHODS

Animals

Adult male mice of different strains were used in this study: CBA/N and C57BL/6J wild type (WT) (purchased from Janvier Labs, Le Genest-Saint-Isle, France), C57BL/6J with a global knock-out (KO) of Thra, the gene encoding TRα (Thra 0/0 ) or their control littermate (Thra +/+ ) [START_REF] Gauthier | Genetic analysis reveals different functions for the products of the thyroid hormone receptor alpha locus[END_REF] and C57BL/6J with a neuronal-specific expression of TRα1 L400R , a dominant negative form of TRα (Cre3xThra AMI/+ ) or their control littermates (Thra AMI/+ ) [START_REF] Quignodon | A point mutation in the activation function 2 domain of thyroid hormone receptor alpha1 expressed after CRE-mediated recombination partially recapitulates hypothyroidism[END_REF][START_REF] Richard | Neuronal expression of a thyroid hormone receptor α mutation alters mouse behaviour[END_REF], both provided by the Institut de Génomique Fonctionnelle de Lyon. The Cre3 mouse line [START_REF] Banares | Novel panneuronal Cre-transgenic line for conditional ablation of genes in the nervous system[END_REF] presents Cre expression and activity in neurons but not in glial cells, and the percentage of neurons expressing Cre depends on the brain areas with a particularly high level in the hypothalamus [START_REF] Richard | Neuronal expression of a thyroid hormone receptor α mutation alters mouse behaviour[END_REF]. In the Cre3xThra AMI/+ mice, the Thra modification induces the expression of the TRα1 L400R mutant receptor, and despite the persistence of an intact Thra allele, the dominant-negative influence of TRα1 L400R eliminates any capacity of the cells to respond to T3 [START_REF] Quignodon | A point mutation in the activation function 2 domain of thyroid hormone receptor alpha1 expressed after CRE-mediated recombination partially recapitulates hypothyroidism[END_REF]. In order to verify the phenotype of the mutated mice, we performed dual immunohistochemistry of TRα and RFPR-3 in two Cre3xThra AMI/+ and observed, as expected, a strikingly low expression of TRα in the mediobasal hypothalamus and no colocalisation of TRα with RFRP-3 (data not shown).

Mice were housed in approved type II cages with food and water ad libitum, at 20°C ± 1°C, under 12 hours white light/ 12 hours red light (12h L/12h D) photoperiod for at least one week before experimentation. The first experiment investigated the effect of photoperiod in melatonin proficient and deficient mice: CBA/N and C57BL/6J WT were kept in LP (16h L/ 8h D) or SP (8h L/16h D) for 6 weeks (n=6 for each of the 4 groups). The second experiment investigated the effect of nighttime exposure to exogenous melatonin in mice mutated for TRα (Thra 0/0 and Cre3xThra AMI/+ ) or their negative littermates (Thra +/+ and Thra AMI/+ ): mice (n=5-7 per group) were placed in SP (10h L/14h D; here SP night was reduced to 14h to obtain a melatonin peak duration similar to that observed in 8h L/16h D adapted Syrian hamsters and melatonin proficient mice (27, 28)) and were given drinking water containing either melatonin (80 mg/L in 0.1% ethanol) or vehicle (0.1% ethanol) during the night, and pure water during the day for at least 6 weeks. This protocol allowed a 4 fold higher nighttime circulating melatonin concentration in mice drinking melatonin as compared to mice drinking vehicle (337,89 pg/ml versus 85,97 pg/ml, p=0.0386, measured by radioimmunoassay [START_REF] Bothorel | In the rat, exogenous melatonin increases the amplitude of pineal melatonin secretion by a direct action on the circadian clock[END_REF]). Although our preliminary data confirmed those of Ono et al [START_REF] Ono | Involvement of thyrotropin in photoperiodic signal transduction in mice[END_REF] that an acute injection of melatonin per day is sufficient to trigger a response in the PT and hypothalamus (data not shown), we decided to perform the long term melatonin administration in the drinking water for

Tissue sampling

At the end of the experiment, animals were euthanized in the morning (from 1 h to 4 h after lights on) by CO2 inhalation. Body weight was measured and testis were dissected and weighted, animals were then transcardiacally perfused first with 10 mL phosphate buffer saline (PBS) then 20 mL periodate-lysineparaformaldéhyde fixative solution [START_REF] Mclean | Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy[END_REF]. Brains were extracted, conserved in the fixative solution for 12 h then dehydrated through an ethanol gradient and embedded in polyethylene glycol [START_REF] Klosen | PEG embedding for immunocytochemistry: application to the analysis of immunoreactivity loss during histological processing[END_REF]. Serial 12 µm-thin coronal sections were made with a microtome from the preoptic area to the posterior hypothalamus. For each animal, serial sections were dispatched on 10 SuperFrost® Plus slides (Thermo Scientific, MA, USA) in order to have 14 to 18 sections spanning the area of interest per slide. Brain sections were dried 15 min at 60°C and stored either at -80°C for in situ hybridization or at 4°C in Tris buffer saline (TBS) with 0.02% sodium azide (NaN3) for immunohistochemistry.

Non-radioactive in situ hybridization

In situ hybridization was performed with 300 to 400 nucleotide-long antisense riboprobes selective for mouse Tshb (NM_009432.2), Dio2 (NM_010050.4), Dio3 (NM_172119.2), Rfrp (NM_021892.1) and rat kiss1 (NM_181692.1) validated on mouse tissue and labeled with digoxigenin (DIG RNA labeling kit, Roche, Meylan, France) according to Klosen et al, 2013 (14). Briefly, brain sections were post-fixed in formaldehyde 4% in phosphate buffer (PB) for 10 min at room temperature, washed with PBS, treated by 0.5 μg/mL proteinase K (Roche) for 30 min at 37°C, washed with cold PBS, post-fixed in formaldehyde 2% in PB, washed in PBS + 0.01% diethyl pyrocarbonate (DEPC), acetylated twice for 10 min in 100 mM triethanolamine with respectively 0.25% and 0.5% acetic anhydride, washed with PBS + 0.01% DEPC, equilibrated in 5X saline sodium citrate (SSC) + 0.05% tween 20 + 0.01% DEPC twice for 5 min at room temperature, and finally incubated for 48 h at 60°C with 1% (about 200ng/ml) riboprobes in a hybridization buffer containing 50% formamide + 5X SSC + 5X denhart solution + 1 mg/ml salmon DNA + 0.1% tween 20 + 0.04% DEPC. Slides were then washed once in 5X SCC + 0.05% tween 20 + 0.01% DEPC buffer, and post-hybridization was performed at high stringency to reduce non-specific labeling in a 0.01X SSC + 0.05% tween 20 solution at 72°C for 6 x 10 min. Slides were washed once in A-DIG + 0.05% tween 20 solution, incubated with blocking buffer (Roche) + 0.02% azide (BBT) for 1 h, and riboprobe labelling was detected by an overnight incubation with an anti-digoxigenin antibody coupled to alkaline phosphatase (AP, Roche; 1/5000 in BBT). Thereafter, slides were washed once in A-DIG + 0.05% tween 20 solution then equilibrated in AP buffer. AP activity was visualized with a mixture of nitro blue tetrazolium (NBT)/ bromo-chloro-indolyl phosphate (BCIP) in AP buffer for 1 to 7 h depending on the probes and stopped with pure water before the staining intensity reached saturation. Slides were then covered with cristal mount premounting media (Sigma Aldrich, MO, USA) and coverslipped with Eukit (Sigma Aldrich).

Immunohistochemistry

The Tshb mRNA level being very low in the C57BL/6J mice pars tuberalis, we performed an immunochemical detection of the TSHβ protein to study melatonin-dependent variations of TSHβ expression in these mouse strains, given the tight correlation between Tshb mRNA and protein levels [START_REF] Dardente | MT1 melatonin receptor mRNA expressing cells in the pars tuberalis of the European hamster: effect of photoperiod[END_REF].

Immunolabelling of TSHβ was done at room temperature using an avidine-biotin system of detection. Brain sections were pretreated with a blocking reagent (TBS + 3% powder milk + 0.02% NaN3) for 1 h, incubated overnight with the primary TSHβ antibody (rabbit anti-TSHβ antibody; National Hormone and Pituitary Program AFP-12747891; diluted at 1/65 000 in TBS + 0.05% tween 20 + 1% donkey serum), washed in TBS + 0.02% tween 20, and finally incubated with a biotinylated secondary antibody (donkey anti-rabbit bio; Jackson ImmunoResearch, PA, USA; diluted at 1/2 000 in TBS + 0.05% tween 20 + 1% donkey serum) for 1 h. After washing in TBS + 0.02% tween 20, the signal was amplified by a 1 h incubation of neutravidine-horseradish peroxydase solution (HRP; Thermo Scientific; 1/2 000 in TBS + 0.02% tween 20 + 0.2% cold water fish gelatin). Sections were washed twice in TBS + 0.02% tween 20, then once in Tris-Imidazole buffer (TBI) buffer and then HRP activity was detected with a 30 min incubation in 1% diaminobenzidine (DAB) + 0.003% H 2 O 2 in TBI buffer. Finally, brain sections were dehydrated by 5 min incubation in solutions of increasing alcohol concentration (from 70 to 100%), and coverslipped with Eukit.

In order to look for TRα localization in tanycytes, we performed dual immunochemical labelling of TRα with the tanycyte marker vimentin in C57BL/6J mice. Brain sections were first treated for an antigen reactivation in citrate buffer (pH 6.0) for 1 h at 95°C, washed twice in PBS, pretreated with the blocking reagent for 1 h, and incubated with the primary TRα antibody (rabbit anti-TRα antibody; tested [START_REF] Dufourny | The Content of Thyroid Hormone Receptor α in Ewe Kisspeptin Neurones is not Season-Dependent[END_REF] and kindly provided by Dr L. Dufourny (INRA, Nouzilly); diluted at 1/250 in TBS + 0.20% triton X100 + 1% donkey serum) for 48 h at 4°C, washed in TBS + 0.02% tween 20, and finally incubated with an Alexa 594 secondary antibody (donkey anti-rabbit Alexa594; Invitrogen, CA, USA; diluted at 1/1 000 in TBS + 0.05% tween 20 + 1% donkey serum) for 1 h. After two washing with TBS, the vimentin labelling was performed following the same protocol than for TSHβ immunolabelling using a vimentin primary antibody (chicken anti-vimentin antibody; Merck, Darmstadt, Germany; diluted at 1/5 000), a biotinylated anti-chicken secondary antibody (anti-chicken bio; Jackson ImmunoResearch; diluted at 1/2 000), and HRP activity detection was done by using hapten-conjugated tyramides (diluted at 1/500 in PBS + 100 mM imidazole + 0.001% H 2 O 2 ). Slides were washed with PBS, covered with cristal mount, and coverslipped with Eukit.

Labeling of both TSHβ and TRα/vimentin was observed with an optic microscope (Leica Microsystems, Rueil-Malmaison, France) equipped with a fluorescent lamp (Leica Microsystems) to visualize fluorescent staining.

Data analysis and statistics

For each gene/protein investigated, brain sections of all animals of a given experimental group were processed similarly (treatments for labeling, optical microscope setting and analysis). Signal quantification was done by a person unaware of the animal's experimental condition.

For the quantification of Kiss1 and Rfrp expression, the total number of labeled neurons in all sections of an individual hypothalamus was counted manually with an optic microscope (Leica Microsystems), equipped with an Olympus camera (Olympus France, Rungis, France, x20). For each animal, data is given as the total number of counted labelled neurons. For the quantification of Tshb, Dio2 and Dio3, the intensity of the signal was quantified with the ImageJ software (W. S. Rasband, U.S. National Institutes of Health, Bethesda, MD, USA), on pictures taken with an optic microscope equipped with camera (Leica Microsystems, x10). All pictures for a given gene were taken with the same microscope and camera setting parameters and a background image without section was taken for each slide and subtracted to overcome any irregularities of illumination of the picture field. Signal intensity was measured by calculating the mean pixel grey value (arbitrary unit) of a line covering the entire pars tuberalis for Tshb, and the β and α tanycytes for Dio2 and Dio3 respectively. For each animal, data results from the mean of signal intensity measured on 3 selected sections of the area of interest.

All data are presented as mean ± SEM of 4 to 7 animals per experimental group. Graphs and statistical analysis were done with GraphPad 6.0 (GraphPad software Inc., San Diego, CA, USA). Variance homogeneity and normality were tested and an adapted Mann-Whitney non-parametric test was performed to analyze the mean differences of expression for each gene and a non-parametric Spearman test was performed to analyze the correlation between Dio2 and Rfrp expression.

RESULTS

CBA

Differential photoperiodic gene regulation between melatonin-proficient (CBA/N) and melatonin-deficient (C57BL/6J) mice

In order to assess how photoperiod regulates expression of Tshb in the pars tuberalis, Dio2 and Dio3 in the tanycytes, and Rfrp and Kiss1 in the hypothalamus of both melatonin proficient and melatonin deficient mice, both CBA/N and C57BL/6J were exposed to either LP (16L/8D) or SP (8L/16D) conditions for 6 weeks. SP exposed CBA/N mice exhibited robust inhibition of Tshb, Dio2, and Rfrp expression, and induction of Dio3 expression as compared to LP exposed CBA/N mice (Figure 1A). In contrast, in the C57BL/6J mice, none of these genes showed photoperiodic change in their expression (Figure 1B). Moreover, in CBA/N mice Dio2 expression is strongly correlated to the number of Rfrp expressing neurons whereas there is no such correlation in C57BL/6J mice (Figure 1A andB). No photoperiodic change in Kiss1 expression and the weight of body, testis, and seminal vesicles has been observed in the two mouse strains (data not shown).

Exogenous melatonin restores SP inhibition of Rfrp expression in control

Thra +/+ but not Thra 0/0 C57BL/6J mice In order to investigate whether melatonin is able to regulate pituitary and hypothalamic gene expression in C57BL/6J mice, exogenous melatonin or vehicle were provided in the drinking water during the dark period.

Nighttime melatonin induced a significant decrease in TSHβ, Dio2 and Rfrp expression and a significant increase in Dio3 expression (Figure 2A) therefore mimicking the effect of SP in melatonin proficient CBA/N mice (Figure 1A). To assess whether this melatonin signal integration requires functional TRα, a similar melatonin supplementation was given to mice bearing a complete mutation of TRα. In Thra 0/0 mice, melatonin still reduced TSHβ and Dio2 and increased Dio3 expression but was no longer able to inhibit Rfrp expression (Figure 2B). Moreover, while there was a significant correlation between Dio2 and the number of Rfrp expressing neurons in the C57BL/6J WT mice, this correlation no longer existed in the Thra 0/0 mice (Figures 2A andB). This result demonstrates the key role of TRα in mediating the melatonin-dependent thyroid hormone effects on Rfrp expression. As previously observed for the photoperiod experiment, melatonin administration had no effect on Kiss1 expression and on the weight of body, testis, and seminal vesicles in the two mouse strains (data not shown). Of note however, and in agreement with earlier observations [START_REF] Guadaño-Ferraz | Lack of thyroid hormone receptor alpha1 is associated with selective alterations in behavior and hippocampal circuits[END_REF], Thra 0/0 mice appeared more anxious and slightly smaller (body weight = 24.6 ± 2.7g, n = 13) than their control littermate (body weight = 26.2 ± 4.1 g, n = 13; Student t-test p = 0.0409).

Melatonin inhibits Rfrp expression through non-neuronal TRα

As TRα is expressed by various cell types in the nervous system [START_REF] Wallis | The thyroid hormone receptor alpha1 protein is expressed in embryonic postmitotic neurons and persists in most adult neurons[END_REF] carrying a selective mutation of Thra in neurons (Cre3xThra AMI/+ ) or their control littermate (Thra AMI/+ ). In both groups, melatonin similarly regulated TSHβ, Dio2/3 but also Rfrp expression, independently of the presence (Figure 3A) or absence (Figure 3B) of neuronal TRα. Moreover, both groups kept a significant correlation between the expression of Dio2 and the number of Rfrp expressing neurons (Figure 3 A andB) therefore indicating a conserved link between Dio2 and Rfrp expression with or without neuronal TRα Considering the non-neuronal effect of TRα on Rfrp expression, we investigated whether TRα could be expressed in the tanycytes, visualized by a specific vimentin labelling. The dual labelling of TRα and vimentin showed a strong co-localization of both proteins, suggesting that tanycytes could integrate the T3 signal through TRα receptors (Figure 3C).

Discussion

Since the discovery of the fundamental role of hypothalamic T3 in relaying seasonal cues to the central nervous system [START_REF] Yoshimura | Lightinduced hormone conversion of T4 to T3 regulates photoperiodic response of gonads in birds[END_REF], numerous studies have sought to understand the neuroendocrine pathways linking the annual changes in photoperiod with the central reproductive network. In mammals, melatonin is key to this adaptive process since it drives the seasonal changes in the pars tuberalis TSH [START_REF] Hanon | Ancestral TSH mechanism signals summer in a photoperiodic mammal[END_REF][START_REF] Wittkowski | Photoperiod-dependent changes in TSH-like immunoreactivity of cells in the hypophysial pars tuberalis of the Djungarian hamster, Phodopus sungorus[END_REF] which in turn regulates hypothalamic T3 via a switch in the tanycytic Dio2/Dio3 ratio [START_REF] Dardente | A molecular switch for photoperiod responsiveness in mammals[END_REF][START_REF] Yasuo | The reciprocal switching of two thyroid hormone-activating and -inactivating enzyme genes is involved in the photoperiodic gonadal response of Japanese quail[END_REF][START_REF] Nishiwaki-Ohkawa | Molecular basis for regulating seasonal reproduction in vertebrates[END_REF]. Further studies then demonstrated that the photoperiodic change in the TSH/T3 signal drive seasonal reproduction through a specific action on Kp and/or RFRP-3 neurons known to regulate GnRH neuronal activity [START_REF] Klosen | TSH restores a summer phenotype in photoinhibited mammals via the RF-amides RFRP3 and kisspeptin[END_REF][START_REF] Angelopoulou | Functional Implications of RFRP-3 in the Central Control of Daily and Seasonal Rhythms in Reproduction[END_REF][START_REF] Simonneaux | A Kiss to drive rhythms in reproduction[END_REF][START_REF] Henson | Exogenous T₃ elicits long day-like alterations in testis size and the RFamides Kisspeptin and gonadotropin-inhibitory hormone in short-day Siberian hamsters[END_REF][START_REF] Freeman | Exogenous T3 mimics long day lengths in Siberian hamsters[END_REF].

Despite these major advances in seasonal reproductive physiology, our understanding of the underlying molecular mechanisms involved has been hampered by the lack of relevant genetically modified seasonal animal models.

In this study we show that mice, although showing no overt seasonal phenotype, can be used to investigate the molecular pathway involved in the melatonin signal integration into the central nervous system. Thus, we reveal that melatonin-proficient CBA/N mice, but not melatonin-deficient C57BL/6J mice, exhibit a significant photoperiodic variations in the pars tuberalis Tshb, tanycytic Dio2 and Dio3 gene expression (as already reported [START_REF] Ono | Involvement of thyrotropin in photoperiodic signal transduction in mice[END_REF]), and also a significant SP-induced reduction in the number of neurons expressing Rfrp gene. By contrast, however, Kiss1 expression does not show photoperiodic variation in CBA/N mice which is in line with the absence of overt photoperiodic changes in their reproductive activity. Altogether, these data indicate that in the melatonin-proficient CBA/N mice, the photoperiodic signal carried by melatonin is integrated down to the hypothalamic Rfrp expressing neurons, as reported in all other seasonal species investigated so far [START_REF] Angelopoulou | Functional Implications of RFRP-3 in the Central Control of Daily and Seasonal Rhythms in Reproduction[END_REF], while Kiss1 expression appears unaffected by the environmental photoperiod in mice. Although C57BL/6J mice are deficient in melatonin, they have functional melatonin [START_REF] Yasuo | Melatonin transmits photoperiodic signals through the MT1 melatonin receptor[END_REF] and TSH [START_REF] Ono | Involvement of thyrotropin in photoperiodic signal transduction in mice[END_REF] receptors and an acute injection of exogenous melatonin regulates Tshb and Dio2/Dio3 gene expression. Here, using a protocol mimicking a SP-melatonin pattern [START_REF] Kasahara | Genetic variation of melatonin productivity in laboratory mice under domestication[END_REF][START_REF] Ribelayga | HIOMT drives the photoperiodic changes in the amplitude of the melatonin peak of the Siberian hamster[END_REF] in C57BL/6J mice, we found that the melatonin-induced switch in the Dio2/Dio3 ratio is associated with a significant reduction in the number of Rfrp expressing neurons, as observed in CBA/N mice. These results demonstrate that nighttime melatonin supplementation in C57BL/6J mice is able to restore photoperiodic like variations in the genes involved in seasonal timing, indicating that this animal model is suitable to tackle the molecular mechanisms underlying photoperiodic integration into the rodent hypothalamus. Of note, in C57BL/6J and CBA/N mice, Rfrp response to melatonin appears smaller than that of the other melatonin-driven genes. This might be related to species-dependent seasonal phenotype as Rfrp's inhibition by melatonin (or SP) is larger in hamsters [START_REF] Revel | RFamide-related peptide gene is a melatonin-driven photoperiodic gene[END_REF] and MSM mice [START_REF] Sáenz De Miera | Photoperiodic regulation in a wild-derived mouse strain[END_REF].

Earlier studies reported that central administration of TSH in SP-adapted hamsters restores the LP level of Rfrp expression, suggesting that the number of RFRP-3 expressing neurons is regulated by T3 [START_REF] Klosen | TSH restores a summer phenotype in photoinhibited mammals via the RF-amides RFRP3 and kisspeptin[END_REF]. In order to address this hypothesis, we investigated the effect of nighttime melatonin supplementation in C57BL/6J mice carrying a global Thra knock-out. Melatonin in Thra 0/0 mice is no longer able to inhibit the number of Rfrp expressing neurons while it still regulates Tshb, and Dio2/Dio3 expression. Furthermore, the significant correlation between Dio2 and Rfrp expression observed in melatonin-proficient CBA/N and in melatoninsupplemented C57BL/6J mice is lost in mice lacking TRα. Therefore, the melatonin inhibition of Rfrp expression appears to involve T3 signaling on TRα, whereas the seasonal regulation of Tshb in the pars tuberalis and Dio2/Dio3 in the tanycytes is independent of this T3-TRα pathway.

TRα is widely distributed in the central nervous system [START_REF] Wallis | The thyroid hormone receptor alpha1 protein is expressed in embryonic postmitotic neurons and persists in most adult neurons[END_REF][START_REF] Bradley | Differential expression of alpha and beta thyroid hormone receptor genes in rat brain and pituitary[END_REF][START_REF] Bradley | Spatial and temporal expression of alpha-and beta-thyroid hormone receptor mRNAs, including the beta 2-subtype, in the developing mammalian nervous system[END_REF][START_REF] Bernal | Thyroid hormone transporters--functions and clinical implications[END_REF], especially in the hypothalamic region [START_REF] Dufourny | The Content of Thyroid Hormone Receptor α in Ewe Kisspeptin Neurones is not Season-Dependent[END_REF][START_REF] Herwig | Hypothalamic thyroid hormone in energy balance regulation[END_REF] suggesting that T3 may act directly on RFRP-3 neurons. However, we observed that nighttime melatonin supplementation in C57BL/6J mice lacking TRα signaling in neurons only [START_REF] Quignodon | A point mutation in the activation function 2 domain of thyroid hormone receptor alpha1 expressed after CRE-mediated recombination partially recapitulates hypothyroidism[END_REF] is still able to reduce the number of Rfrp expressing neurons, with a conserved correlation between Dio2 and Rfrp expression, as it does in their control littermates. Preservation of the melatonin inhibition of Rfrp expression in absence of TRα signaling in neurons suggests that T3 is acting through non-neuronal cells expressing TRα. Earlier studies reported that astrocytes express thyroid hormones transporters such as MCT-8 and OATPs [START_REF] Warner | Thyroid hormone and the central control of homeostasis[END_REF] but not TRα [START_REF] Wallis | The thyroid hormone receptor alpha1 protein is expressed in embryonic postmitotic neurons and persists in most adult neurons[END_REF][START_REF] Carlson | Immunofluorescent localization of thyroid hormone receptor isoforms in glial cells of rat brain[END_REF], suggesting that astrocytes do not mediate the T3 regulation of RFRP-3 expressing-neurons. Here, we show that TRα is widely expressed in the vimentin-expressing tanycytes, as already reported by Wallis and collaborators who found that tanycytes are the only GFAP-labelled cells expressing the TRα1 receptor in the mouse hypothalamus [START_REF] Wallis | The thyroid hormone receptor alpha1 protein is expressed in embryonic postmitotic neurons and persists in most adult neurons[END_REF].

Tanycytes are ependymal-like glial cells which play a crucial role as hypothalamic integrators of reproduction and energy metabolism [START_REF] Prevot | The Versatile Tanycyte: A Hypothalamic Integrator of Reproduction and Energy Metabolism[END_REF] and exhibit cellular extensions in the mediobasal hypothalamus, and notably in the area where RFRP-3 neurons are located (52, unpublished observations). Those nonneuronal TRα-expressing cells could therefore indirectly mediate the melatonin-driven regulation of Rfrp expression by delivering a messenger molecule regulated by T3 to the RFRP-3 neurons. Tanycytes are also recognized as a putative niche of stem cells able to divide and create newborn neurons and glial cells invading the neighboring hypothalamus during development but also in adult [START_REF] Kokoeva | Evidence for constitutive neural cell proliferation in the adult murine hypothalamus[END_REF][START_REF] Xu | Neurogenesis in the ependymal layer of the adult rat 3rd ventricle[END_REF][START_REF] Kokoeva | Neurogenesis in the hypothalamus of adult mice: potential role in energy balance[END_REF]. A photoperiodic/seasonal regulation of hypothalamic neurogenesis has been reported in adult Syrian hamster [START_REF] Huang | Photoperiod regulates neuronal bromodeoxyuridine labeling in the brain of a seasonally breeding mammal[END_REF] and sheep [START_REF] Migaud | Seasonal changes in cell proliferation in the adult sheep brain and pars tuberalis[END_REF][START_REF] Hazlerigg | Photoperiodic variation in CD45-positive cells and cell proliferation in the mediobasal hypothalamus of the Soay sheep[END_REF], which could originate in the tanycytes [START_REF] Prevot | The Versatile Tanycyte: A Hypothalamic Integrator of Reproduction and Energy Metabolism[END_REF][START_REF] Batailler | Pineal-dependent increase of hypothalamic neurogenesis contributes to the timing of seasonal reproduction in sheep[END_REF][START_REF] Helfer | A unifying hypothesis for control of body weight and reproduction in seasonally breeding mammals[END_REF]. Notably, blocking seasonal neurogenesis in sheep has been demonstrated to alter the timing of annual reproduction [START_REF] Batailler | Pineal-dependent increase of hypothalamic neurogenesis contributes to the timing of seasonal reproduction in sheep[END_REF]. Since T3 is involved in adult neurogenesis [START_REF] Gomes | Cerebellar astrocytes treated by thyroid hormone modulate neuronal proliferation[END_REF][START_REF] Kapoor | Perspectives on thyroid hormone action in adult neurogenesis[END_REF], we can hypothesize that disruption of the tanycytic TRα signaling could impair hypothalamic neurogenesis, which in turn would prevent photoperiodic regulation of the number of RFRP-3 neurons.

In conclusion, our results have established that endogenous or exogenous nighttime melatonin not only regulates TSHβ and Dio2/Dio3, but also the number of Rfrp expressing neurons in mice. Further, using genetically modified C57BL/6J mice, we have demonstrated the pivotal role of T3 signaling through TRα in the melatonin-driven regulation of Rfrp neurons. Finally, because the selective inactivation of TRα signaling in neurons does not impair Rfrp regulation by melatonin, we hypothesized that T3 may act through the tanycytes, which express a high level of TRα. Considering the role of hypothalamic neurogenesis in the seasonal reproduction [START_REF] Kokoeva | Neurogenesis in the hypothalamus of adult mice: potential role in energy balance[END_REF], future studies should now investigate whether the melatonin-driven regulation of RFRP-3 neurons involves neurogenesis processes initiated by a T3 action on the tanycytes. expression and the number of Rfrp neurons in C57BL/6J Thra +/+ control mice (r = 0.6025; r 2 =0.3630; p = 0.0323) and lack of correlation in C57BL/6J Thra 0/0 mice (r = 0.1240; r 2 = 00154; p = 0.,6852).

  physiological and practical reasons. Experiments were conducted in accordance with the French National Law implementing the European Communities Council Directive 2010/63/EU and the French Directive 2013-118. All procedures were validated by the Animal Welfare Committee of the Animal Resource and Experimentation platform (Chronobiotron UMS 3415) of the Strasbourg Institute of Neuroscience.

RR

  b m R N A (a .u .) fr p m R N A (c e lls /a n im a l) D io 2 m R N A (a .u .) fr p m R N A (c e lls /a n im a l) D io 2 m R N A (a .u .) p = 0 .2 6 6 2

5 R 5 RR 5 R

 555 , we further investigated whether TRαexpressing neurons mediate the inhibitory effect of melatonin on Rfrp expression in the DMH RFRP-3 neurons. Nighttime melatonin supplementation in drinking water was thus provided in C57BL/6J mice C57BL/6J Thra +/fr p m R N A (c e lls /a n im a l) D io 2 m R N A (a .u .) fr p m R N A (c e lls /a n im a l) D io 2 m R N A (a .u .) fr p m R N A (c e lls /a n im a l) D io 2 m R N A (a .u .) fr p m R N A (c e lls /a n im a l) D io 2 m R N A (a .u .)

Figure 1 :

 1 Figure 1: Effect of photoperiod on Tshb in the pars tuberalis, Dio2 and Dio3 in the tanycytes, and Rfrp in the dorsomedial hypothalamus of melatonin proficient CBA/N and melatonin deficient C57BL/6J mice. CBA/N (A) and C57BL/6J (B) adult male mice were exposed to either long (LP: 16h light/8h dark, white bars) or short (SP: 8h light/16h dark, black bars) photoperiod for 6 weeks. Expression of TSHβ, Dio2, Dio3 and Rfrp were assessed by non-radioactive in situ hybridization. TSHβ, Dio2 and Dio3 values are given as mean level of grey value in arbitrary unit (a.u.), and Rfrp value is given as total number of labelled cells counted per animal. Data are given as individual values and mean values ±SEM of 5-6 mice; * p< 0,05 ; ** p<0,01. Right panels are scatterplot showing the positive correlation between Dio2 expression and the number of Rfrp neurons in CBA/N mice (r = 0.7576; r 2 = 0.5740; p = 0.0149) and lack of correlation in C57BL/6J mice (r = -0.3497; r 2 = 0.1223; p = 0.2662). Representative photomicrographs of in situ hybridization for Tshb, Dio2, Dio3 and Rfrp in SP an LP CBA/N and C57BL/6J are presented under each corresponding plot. Scale bars = 75µm.

Figure 2 :

 2 Figure 2: Effect of exogenous melatonin on TSHβ in the pars tuberalis, Dio2 and Dio3 in the tanycytes and Rfrp in the dorsomedial hypothalamus in C57BL/6J mice with (Thra 0/0 ) or without (Thra +/+ ) global TRα mutation. C57BL/6J Thra +/+ control (A) and Thra 0/0 (B) adult male mice, kept in short photoperiod (SP: 10h light/14h dark), were given either vehicle (0.1% ethanol in water, plain bars) or melatonin (80 mg/L melatonin, 0.1% ethanol in water, gridded bars) in the drinking water during the dark period and normal water during the light period for at least 6 weeks. Expression of TSHβ was assessed by immunohistochemistry and Dio2, Dio3 and Rfrp by non-radioactive in situ hybridization. TSHβ, Dio2 and Dio3 values are given as mean level of grey value in arbitrary unit (a.u.), and Rfrp value is given as total number of labelled cells counted per animal. Data are given as individual values and mean values ±SEM of 6-7 mice; * p< 0.05 ; ** p<0.01. Right panels are scatterplot showing the positive correlation between Dio2

Figure 3 :

 3 Figure 3: Effect of exogenous melatonin on TSHβ in the pars tuberalis, Dio2 and Dio3 in the tanycytes and Rfrp in the dorsomedial hypothalamus in C57BL/6J mice with (Cre3xThra AMI/+ ) or without (Thra AMI/+ control) selective neuronal TRα mutation, and dual immunolabelling of TRα with the tanycytic marker vimentin in C57BL/6J mice. C57BL/6J Thra AMI/+ control (A) and Cre3xThra AMI/+ (B), adult male mice kept in short (SP: 10h light/14h dark) photoperiod, were given either vehicle (0.1% ethanol in water, plain bars) or melatonin (80 mg/L melatonin, 0.1% ethanol in water, gridded bars) in the drinking water during the dark period and normal water during the light period for at least 6 weeks. Expression of TSHβ was assessed by immunochemistry and expression of Dio2, Dio3 and Rfrp by non-radioactive in situ hybridization. TSHβ, Dio2 and Dio3 values are given as level of labelling intensity in the region of interest

Acknowledgements

We thank all the staff of the Animal Resource and Experimentation platform of Strasbourg (Chronobiotron) for their help with animal care, Laurence Dufourny and Marion Millet for the generous gift of antibodies and Béatrice Bothorel for the melatonin assay.

Author contributions

C. Quignon designed, performed research and wrote the manuscript, M. Beymer provided probes and CBA/N tissue, K. Gauthier provided the C57BL/6J Thra 0/0 , Thra +/+ , Cre3xThra AMI/+ and Thra AMI/+ , F. Gauer discussed data significance, and V. Simonneaux designed the experimental studies, discussed data significance, and corrected the manuscript. All co-authors have read and approved the manuscript. in arbitrary unit (a.u.), and Rfrp value is given as total number of labelled cells counted per animal. Values are given as mean ±SEM of 4-7 mice; * p< 0.05 ; ** p<0.01. Right panels are scatterplot showing the positive correlation between Dio2 expression and the number of Rfrp neurons in C57BL/6J Thra AMI/+ control mice (r = 0.7123; r 2 =0.5074; p = 0.0323) and in C57BL/6J Cre3xThra AMI/+ mice (r = 0.8571; r 2 = 0.7346; p = 0.6852). (C) Photomicrographs of an immunohistochemical detection of vimentin detected with tyramide (green) and TRα detected with Alexa594 (red) in the tanycyte with the co-labelling shown on the merge image; Scale bar = 50µm.


Ekaterina Mamontova

Philippe Trens

Fabrice Salles

Bernard Fraisse

Olinda Carreon Gimello

Yannick Guari

Joulia Larionova

Jérôme Long

Enantioselective separation under humid conditions by chiral Hofmann clathrates: new opportunities for vintage materials

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Chirality is a matter of critical importance in many areas such as biology, 1 pharmaceutical industry, 2 agriculture 3 and catalysis. 4 Several molecules emblematic of life on Earth, such as amino acids or carbohydrates, exist in both, right and left handed shape. However, biological life has a marked preference for only one enantiomer (phenomenon called a homochirality), which cannot be superposed on its mirror image. [START_REF] Plaxco | Astrobiology: a brief introduction[END_REF] Moreover, most part of biological processes involving metabolites and enzymes usually show a high degree of specificity toward only one enantiomer. [START_REF] Bentley | [END_REF] Right and left handed enantiomers can exhibit large differences in their behavior in the living organisms in terms of recognition by receptors, physiological activity, absorption, distribution and clearance, potency, metabolism and toxicology. In this sense, a large percentage of pharmaceutical agents and drugs are optically active and therefore specifically interact through chiral recognition. [7][8][9] Following the Food and Drug Administration (FDA) statement, while some drug enantiomers present similar pharmacological effect, such as ibuprofen, others might have completely antagonist harmful activities. [START_REF] Bentley | [END_REF] Hence, the separation of enantiomers is decisive for both, economic and health concerns. [7][8][9][10] Regardless the targeted application, chiral recognition is a necessary condition to ensure the efficiency of the enantiomers' separation. In the industry, various processes allowing the separation of enantiomers using chiral stationary phases have been developed starting from a procedure applied in early 1970's involving the immobilization of optically active small molecules on silica gels or organic polymers. 11 This method is still widely used in separation chromatography but the rather recent developments made this functionalization of lesser interest. [12][13] Amongst these, bio-sourced, synthetic imprinted or helical polymers proved to be successful, even though their synthesis was not straightforward. [14][15] However, the mobile phase is usually difficult to define for a specific bio-polymer and each system therefore requires a long term optimization due to the complexity of the supported functionalities. Furthermore, the specific surface area was not considered as a primary parameter in these materials and, hence, developing the internal surface appears highly important to increase the efficiency of the separation. In this sense, microporous chiral materials exhibiting a permanent porosity could be therefore viewed as promising systems for enantioselective separation. Nevertheless, the design of mechanically and thermally robust inorganic chiral frameworks, such as zeolites, is highly challenging, although some approaches involving the synthesis with chiral directing or inductive agents have already been proposed. 16 Moreover, the size and the topology of the zeolite's pores may be difficult to adjust towards specific guests as, for instance, large organic molecules found in the pharmaceutics. Following this idea, chiral and porous molecule-based materials, such as Metal-Organic Frameworks (MOF) are promising for such purpose owing to their flexible molecular structures, adjustable pores' size/shape and chemical affinity towards targeted guest molecule. 17-20 Different synthetic strategies have been employed to design such chiral MOF consisting in the spontaneous resolution starting from achiral precursors, [21][22] use of chiral templating or induction by chiral agents 7,[23][24] and direct synthesis with enantiopure building blocks. 4,[25][26] While the two former strategies may suffer from predictability due to hardly controllable factors, the latest one often requires expensive or complex building blocks that may limit the synthesis at the multigram scale. Another appealing approach consists in the post-synthetic modification (PSM) of achiral architectures using a large variety of enantiopure molecules that, in addition, allows the specific tailoring of the chemical affinity for a targeted application. 23,[27][28] Furthermore, the easiness of the synthesis, as well as the low cost of some enantiopure molecules constitute an interesting asset. Yet, this strategy requires the presence of reactive or coordinatively unsaturated sites (CUS) in order to ensure a strong chemical interaction between the framework and the grafted chiral species. On the other hand, the aforementioned targeted applications frequently necessitate performing such enantioselective separation in the presence of water at different stages, which could be of critical importance for the separation of some hydrosoluble racemic compounds, such as drugs. This therefore requires the use of materials with a strong robustness towards water, liquid or as vapor. Additionally, liquid separations could be performed above room temperature, which addresses also the hydrothermal stability of these materials. Considerable efforts have recently been made in the family of chiral MOFs in this connection and several examples of hydrostable chiral MOFs have recently been reported, [29][30][31][32] but such systems remain scarce. Moreover and to our knowledge, the investigations on the enantioselective separation in humid conditions have never been achieved using chiral MOF. In this line of thought, Hofmann Clathrates represent a promising alternative to MOF because they present all the aforementioned advantages of MOFs, while exhibiting an exceptional hydrothermal stability. 33 with two trans ligands, which can be substituted to functionalize the coordination network. For instance, a vast variety of bidentate bridging ligands has been used to obtain 3D-pillared Hofmann clathrates for spin-crossover [34][35][36][37][38] or gas adsorption studies. [39][40][41] In this article, we report an elegant strategy to design new chiral Hoffmann Clathrates by the post-synthetic functionalization of a two-dimensional cyano-bridged compound [Ni(H2O)2][Ni(CN)4]•1.5H2O by a simple and affordable enantiopure R or Sphenylethylamine ligands and demonstrate the enantioselective adsorption of butan-2-ol and the separation of its racemic mixture in humid conditions.

Results and Discussions

Synthesis and characterization

The PSM to implement chirality relies on the use of a well-known two-dimensional compound [Ni(H2O)2][Ni(CN)4]•1.5H2O (1), which is one of the benchmark Hofmann clathrates (Fig. 1). [42][43][44] The water molecules occupying the axial coordination sites could be easily removed to generate CUS. [39][40][41] Thus, activation of 1 under vacuum at 130 °C followed by the addition of 2 equivalents of either R, S, or racemic (+/) phenylethylamine (L, Scheme 1) in chloroform affords the formation of light purple solids for R-1, S-1 and light blue solid for (+/)-1, respectively (Fig. S1). The amine functions of L ensure their coordination with the octahedral Ni 2+ sites. The presence of L in the materials is confirmed by the FT-IR spectra, which exhibit its characteristic bands in the 400 -1600 cm -1 spectral window in addition to the cyanide (Ni II-CN-Ni II ) stretching located at 2166 cm 1 for all functionalized compounds (Fig. S2-S3). This latter value is slightly shifted to lower wavenumbers with respect to the pristine compound 1 (( ). We note that in all cases, it was not possible to achieve a full saturation of the CUS that would correspond to two L moieties per octahedral Ni 2+ ions. Besides, a lower ligand content is observed when the reaction is performed with the racemic mixture of L, suggesting a different organization of the structures depending on the chirality of the phenylethylamine. TGA analysis (Fig. S4) shows two distinct weight losses below 190 °C, which could be imputed to interstitial and coordinated water molecules, respectively. While 1 is thermally stable up to 350 °C, TGA curves of R-1, S-1 and (+/)-1 show that the phenylethylamine is lost in the temperature range 200-300 °C for the postfunctionalized clathrates, which remains slightly higher than its boiling point (i.e. 187 °C) due to its stabilization through coordination to the metal-cyanide network. Further evidence for the incorporation of the chiral ligands into the structure could be obtained using solid-state Circular Dichroism (CD) spectroscopy. While the functionalized racemic (+/)-1 clathrate does not show any optical activity, R-1 and S-1 exhibit mirrorsymmetrical dichroism with Cotton effects of opposite signs at max = 310, 355 and 575 nm confirming their enantiomeric relationship (Fig. 2). The broad band at around 575 nm could be ascribed to the d-d transitions of the octahedral Ni 2+ observed in comparable clathrates, [45][46] while the bands in the UV domain correspond to charge transfers of the [Ni(CN)4] 2 and L moieties. These results confirm: (i) the coordination of the chiral ligand to the octahedral Ni 2+ , and (ii) the observed chirality transfer from L to the host-structure. Insights into the structural changes generated by the PSM could be obtained by Powder X-Ray Diffraction (XRPD) analysis. The XRPD pattern and the Le Bail refinement for the pristine material 1 are consistent with the previously published phase crystallizing in the orthorhombic Ima2 space group (Fig. 3, Fig. S5, Table S1). 42 Upon functionalization with L, the pattern was largely changed. While the functionalization with either the enantiopure R or S-phenylethylamine generates comparable PXRD patterns, the use of the racemic phenylethylamine yields a different diffractogram (Fig. 3). Le Bail refinements (Fig. S5, Table S1) indicate that R-1 and S-1 crystallize in the non-centrosymmetric monoclinic space group P2, which is in full agreement with the results obtained from CD. Although, the racemic clathrate (+/)-1 pattern could be refined either in the P2 or P2/m space groups due to identical extinction rules, the absence of dichroic signals rather suggests a crystallization in the centrosymmetric P2/m space group (Table S1). To go further, theoretical modelling was performed in link with PXRD patterns to propose the best models for R-1 and S-1. Using DFT calculations, two different organizations of the phenylethylamine ligands have been tested for both L handednesses and the resulting configuration energy was estimated. 2D structures made by layers of [Ni(R/S-L)(H2O)][Ni(CN)4] were considered, employing a simple model in which each octahedral Ni 2+ ion is coordinated by a chiral ligand L and a water molecule located in trans fashion. It turns out that the lowest energy value consists in the regular alignment of the chiral ligands and water molecules (Fig. 1b) in contrast to an alternate sequence (Fig. S6, Table S2). Such fact could be rationalized by the presence of strong intralayers - interactions involving the phenyl groups of L and cyano ones from [Ni(CN)4] 2 moieties (Fig. S7). Additionally and to probe the occurence of interlayers hydrogen bonds and - a structure constituted by pair of layers with an aligned arrangement ligands was investigated also investigated by DFT (Fig. S6). The associated energy is comparable to that obtained previously for the Intensity / arb. units

2  / ° (+/-)-1 S-1 R-1 1 aligned sequence.
Nevertheless, owing to the experimental L content greater than unity (i.e. 1.40 for R-1 and S-1), some octahedral Ni 2+ sites should incorporate two ligands involving that a mixed arrangement is most likely to occur.

Adsorption Studies

Sorption experiments were then performed to evaluate the efficiency of the chiral porous frameworks to induce an enantioselective adsorption. The TGA results already discussed indicate that the solids could be activated at 100 °C to avoid racemisation and under secondary vacuum without any loss other than water molecules. In order to confirm the integrity of the chiral coordination frameworks upon heating, both FT-IR and CD spectroscopies were carried out on the solid R-1 after such activation process. While the FT-IR spectrum (Fig. S8) is identical with respect to that of the pristine material, the CD clearly confirms that the chirality is retained upon the activation procedure (Fig. S9). Firstly, the sorption features of the racemic adsorbent clathrate (+/)-1 were studied using achiral H2O and racemic (+/)butan-2-ol (referred as (+/)-BuOH) probes. The water sorption isotherms (Fig. S10) exhibit a sigmoidal shape, indicating a switch from hydrophobic to hydrophilic character, as we previously evidenced in the case of related Prussian blue analogues. 47 Namely, this step corresponds to a threshold related to the adsorption of water on the remaining CUS that in turn actuates the hydrophilic character of the framework. In contrast, this step appears for a largely greater relative pressure value when (+/)-BuOH vapour is used and shows a large hysteresis effect upon desorption. For both probes (water and (+/)-BuOH), it could be noticed that the first step is located at a Qads about 0.9 molecules which is close to the expected number of 1.06 CUS based on the previouly established formula. However, the water isotherm shows a continuous increase at high pressure to reach about 3 molecules due its adsorption into the residual porosity. This could be explained by the smaller size of water compared to (+/)-BuOH. The observed water capacity for the racemic solid (+/)-1 is comparable to that of the pristine clathrate 1 (i.e. 2.5 H2O) (Fig. S11). For this later, it could be noticed that higher adsorpion capacities have been observed for instance in the Mn[Ni(CN)4]. 48 Noticeably, the amount of water molecules in such systems remains a complex problem since it influences the crystal structure (three different phases are known). [42][43][44] However it could be noticed that the non-functionalized material 1 shows a step at about a higher 1.8 H2O per unit, in accordance with the two expected CUS in trans position of the octahedral Ni 2+ ions. All these data clearly indicate that despite the PSM, the functionalized clathrates could efficiently adsorb different species in line with the obtained porous volumes based on nitrogen sorption isotherms (Fig. S12). Indeed, the hydrodynamic pore volumes of 1, R-1 and (+/)-1 were found to be 0.315 cm 3 .g 1 , 0.198 cm 3 .g 1 and 0.122 cm 3

.g 1 respectively

To go further, the study of the adsorption of the same sorbates in the chiral functionalized material R-1 was performed (Fig. S13). Remarkably, it could be noticed that the steps of the water isotherms for R-1 and (+/-)-1 solids are located at an identical relative pressure pointing out a comparable hydrophobic/hydrophilic balance despite the slight difference in the L content. The Qads value corresponding to the step for the R-1 adsorbent is found at 0.57, in perfect accordance, with respect to the expected number of 0.60 CUS based on the chemical formula. Since the porous volume are close, this behavior could be at first glance explained by both, the larger amount of coordinated water in (+/)-1 and the different structures of (+/)-1 and R-1.

To our knowledge, enantioselectivity towards an achiral probe has been only recently observed in the MOF Mg2(dobpdc) (dobpdc 4 = 4,4'-dioxidobiphenyl-3,3'-dicarboxylate) functionalized by chiral diamines for CO2 adsorption. 23 Yet, Mg2(dobpdc) crystallizes as racemate (containing equal amount of right and left-handed helices) and the subsequent functionalization induces the formation of diastereomeric domains that alter the non-covalent interactions with CO2. Such phenomenon could however not occur since the pristine clathrate 1 is not a racemate. Hence, the observed differences may be rather ascribed to different structural organizations of (+/)-1 and R-1 as evidenced by the XRD analysis. In addition, the (+/)-BuOH isotherm with R-1 exhibits a striking difference with respect to the racemic (+/)-1 substrate (Fig. S13). Since (+/)-BuOH is composed by an equimolar mixture of R-BuOH and S-BuOH, such behaviour may reflect a chiral recognition effect induced by the formation of different diastereoisomeric domains.

Such scenario is further confirmed by studying the sorption isotherms of the racemic clathrate (+/)-1 with either R-BuOH and S-BuOH chiral probes. To the best of our knowledge, such adsorption using chiral probes has never been performed in MOFs. The obtained sorption isotherms exhibit clear differences depending on the chirality of the sorbate (Fig. 4). Hence, using S-BuOH, the step in the isotherm is found at a lower relative pressure, while the saturation capacity is found 25 % greater with those obtained with R-BuOH. It should be emphasized that enantioselectivity requires an interaction with an active site associated with a chirality. Although, the adsorption capacity (number of sorbates to be accomodated within the framework) could hardly be considered as a proof for enantioselectivity, the location of the step indirectly reflects the affinity of the sorbate for the solid. This suggests the occurrence of enantioselective recognition in our materials mostly caused by diastereoisomeric domains.

To study this phenomenon in details, the adsorption isotherms using both chiral probes (R-BuOH and S-BuOH) and the chiral material R-1 were performed. Firstly, one can notice in Fig. 5 that the adsorption capacities are found weaker with respect to those observed for racemic clatharate (+/)-1. Such effect could be explained by the different chemical formulas of the clathrate that differs from the number of phenylehtylamine ligands. Hence, the weaker number of phenylethylamine (L) in (+/)-1 explains its observed higher capacities with respect to the chiral clathrate. Secondly, Fig. 5 shows the R-BuOH sorption isotherms with R-1 and S-1 clathrates, pointing out the occurrence of distinct features, namely in both the location of the steps and the adsorption capacities. Such behaviour most likely arises from a diastereoisomeric effect obtained by the R-BuOH adsorption generating either R-1@R-BuOH and S-1@R-BuOH pairs that shows different physico-chemical features. To fully corroborate this, the different isotherms of enantiopure and racemic (+/)-BuOH obtained with the chiral R-1 clathrate as a substrate is shown in Fig. 6. One can observe an enantioselective effect depending on the BuOH chirality, in good accordance with the previous experiments. At first glance, the differences in the adsorption capacities may be viewed as less pronounced with respect to those obtained with the racemic clathrate (+/)-1 (Fig. 4). However, it should be stressed that a quantitative comparison between R-1 and (+/)-1 solids could not performed due to the difference in the phenylethylamine content in the materials. Interestingly, the sorption isotherms obtained with the racemic (+/)-BuOH presents an intermediate behaviour: indeed, the step is located at a similar relative pressure value compared to the isotherm obtained with R-BuOH, but its saturation capacity is identical to S-BuOH adsorbate. Moreover, it could be noticed that the R-1 clathrate seems to present a stronger affinity towards S-BuOH based on both, the step location and adsorption capacity (Fig. 6). The conclusions drawn from the analysis of Fig. 5 and6 point out: i) a specific interaction of the racemic (+/)-BuOH towards the chiral clathrate R-1 with respect to the racemic (+/-)-1; ii) a difference in the interaction of R-BuOH and S-BuOH with R-1.

Enantioselective separation

Although, the energy difference in the enantioselective interaction between the and the chiral clathrate is most likely very weak, this chiral recognition effect may be taken as an advantage to perform enantioselective separation in humid conditions, which still remains a challenge in the field of enantiomeric separation using molecule-based materials. To demonstrate this, the clathrate R-1 was used as a chiral stationary phase for performing high-performance liquid chromatography (HPLC) in a short home-made filled column. In light of the significant differences observed in the adsorption isotherms, we selected the same aliphatic alcohol (+/)-BuOH for testing the enantioselective property of our material. Using a mobile acetonitrile/water (70%/30%) phase, the racemic (+/)-BuOH could be separated by R-1 into two clear adjacent peaks (Fig. 7) corresponding to the two constituting enantiomers as identified by the sign of the optical rotation obtained by polarimetry. Thus, S-BuOH is eluted after R-BuOH indicating a preferential interaction of the former with R-1, as suggested by the adsorption isotherms.

R-BuOH R-1 S-1 Q ads / mol.mol -1
Equilibrium relative pressure p/p 0 In our experimental conditions, it was not possible to achieve a better resolution, which might be consistent with the similarity of the adsorption enthalpies. Moreover, we have also performed the chromatographic separation with either pure R-BuOH or S-BuOH (Fig. S14). Although in our conditions, only a minor difference as regards the maximum of the peak could be observed, the S-BuOH chromatogram shows a broader peak suggesting a preferential interaction with R-1. It must be emphasized that the column was simply packed with particles of R-1, which suggests that a better separation could be obtained by using a longer column with homogeneous particles of R-1. Additionally, the injection of the racemic (+/)-BuOH in a classical polar GC column (60 m long, 0.25 mm inner diameter) resulted in a single peak, emphasizing the performance of R-1 for the separation of enantiomers. To our knowledge, this is the first demonstration of such enantioselective separation in humid conditions in a molecule-based material, such as MOF or PCP.

Molecular Simulations

In order to give molecular insights on the enantioselective interaction, DFT calculations have been performed. Firstly, simple models constituted by pairs of either R-BuOH/R-phenylethylamine or S-BuOH/R-phenylethylamine were considered to validate the enantioselective effect. The plausible interactions are given in Fig. S15 and are mainly due to the accessibility of the butan-2ol to approach some parts of the R-phenylethylamine through hydrogen-bonds and C-H/ interactions. It turns out that the difference in the interaction energy between one alcohol molecule and the ligand R-phenylethylamine at the molecular state is close to 2 kJ.mol 1 in favour of S-BuOH. As expected, this weak energy difference is in agreement with an enantioselective effect 49- 50 and is in line with the adsorption results. To go further and explain the observed enantiomeric separation, Monte Carlo simulations were tested to probe the impact of coadsorption, knowing the plausible candidates for the clathrate structures with the parameters given in ESI and the partial charges for the two enantiomers of BuOH (Fig. S16). The adsorption enthalpy values calculated at low loading are slightly different ( 1 kJ.mol 1 ) for R-and S-BuOH in R-1 when mono-adsorption is investigated. Yet, the co-adsorption of the racemic mixture (+/)-BuOH with R-1 shows clearly an enantioselective effect. Hence, difference in the distances of hydrogen-bonds and C-H/ interactions between the alcohol molecules and both chemisorbed water molecules on the Ni 2+ and phenyl rings of the framework (Fig. S17) are observed depending on the BuOH's chirality. As highlighted in the snapshots from the Fig. S16, the same type of interactions is observed between both, R-and S-BuOH enantiomers with the R-1 framework, but the different conformations of the alcohol molecules prevent from obtaining the same magnitude in their strength. This leads to a modification of the adsorption enthalpy values (Table 1), regardless the considered arrangement of the clathrate. Thus, a greater enthalpy adsorption is observed for S-BuOH. This suggests stronger interactions between S-BuOH and R-1, which is in perfect agreement with the chromatography separation results. Hence, this pronounced enantioselective effect when co-adsorption is envisaged highlights the critical role of the competition between R-BuOH and S-BuOH during the adsorption process.

R-()-BuOH

Conclusions

In summary, we have described in this study the first example of post-functionalization to implement chirality in Hofmann clathrates. Hence, reaction of affordable enantiopure amines on the CUS of the two-dimensional clahtrate 1 allows the formation of chiral porous framework accessible at the multigram-scale. Studies of the adsorption isotherms indicate a clear enantioselectivity, which makes these compounds excellent candidates for the chiral separation. Indeed and owing to their great hydrostability, the chiral clathrates are able to separate a racemic mixture of butan-2-ol in humid conditions. Molecular simulations provided microscopic insights into the structural properties and the enantioselective interaction between the chiral clathrates and the butan-2-ol helping to understand this enantioselective preference. This approach could be easily extended to various coordination frameworks of different nature and opens tremendous potentialities for the chiral separation of hydrosoluble compounds.

Experimental Materials and Methods

All chemical reagents were purchased and used without further purification: Nickel(II) nitrate hexahydrate (Alfa Aesar, 98%), Potassium tetracyanonickelate (II) (Aldrich, 99.9%), R-(+)-1-Phenylethylamine (Alfa Aesar, 98%), S-(-)-1-Phenylethylamine (Alfa Aesar, 98%), (+/-)-1-Phenylethylamine (ABCR, 98%), ultra-pure water, Chloroform pure stabilized with ethanol (Carlo Erba).

Synthesis of [Ni(H2O)2][Ni(CN)4]•1.5H2O (1)

The compound was obtained by modified procedure. 39 Following the same procedure for R-1, the racemic compound (+/-)-1 was prepared by addition of the solution of racemic (+/-)-1-Phenylethylamine in chloroform (0.1 M, 180 mL) to the solid 1 (9 mmol, 3.0 g) under argon. The reaction mixture became grey and was stirred under argon overnight. Then, the precipitate was recovered by filtration, washed with chloroform and dried in the air at 60°C. Light blue solid. IR (KBr, cm -1 ): 2166, ν(Ni

Infrared Spectroscopy

Infrared spectra were recorded as KBr disks on a PerkinElmer Spectrum two spectrophotometer.

X-ray Powder Diffraction

X-ray powder diffraction patterns were recorded in the 2 interval 4-60° at room temperature with a PANalytical X'Pert PRO MPD - diffractometer in Bragg-Bentano geometry, with the Cu-K radiation (λ = 1.5418 Å). From each powder diffraction pattern, the unit cell was determined using the Treor and Dicvol programs. Then the cell parameters were refined by the Le Bail method with the Fullprof program using the WinPLOTR interface.

Scanning Electronic Microscopy

Scanning Electronic Microscopy (EDS) analyses were performed on a FEI Quanta FEG 200 instrument. The powders were deposited on an adhesive carbon film and analysed under vacuum. The quantification of the heavy elements was carried out with the INCA software, with a dwell time of 3 µs.

Thermogravimetric Analysis

Thermogravimetric analyses were obtained with a thermal analyser STA 409 Luxx® (Netzsch) in the range 25 -600 °C at heating speed of 5 °C /min.

Circular Dicrhoism

CD measurements were performed on a Jasco J-815 Circular Dichroism Spectropolarimeter at 298 K in solid state (KBr pellets). To prepare pellet for measurements, 1 mg of sample was mixed with 100 mg of KBr. A scan speed of 100 nm.min -1 with data interval of 0.1 nm and bandwidth of 10 nm was used.

Polarimetry

The optical activity measurement was performed on a Polarimeter PerkinElmer Model 341 with Na source lamp with spectral line at 589 nm and Glan-Taylor polarizer.

Adsorption Isotherms

The vapor adsorption/desorption experiments have been performed with a home-made apparatus already described elsewhere. 51 This set-up is based on manometric measurements (with two capacitive pressure gauges (0-10 Torr and 0-1000 Torr). The sample cell can be disconnected from the system to undergo a thermal treatment up to 250°C (depending on the thermal stability of the sample) under a vacuum of 10 3 Torr. The adsorption set-up presented above allows for the choice of the pressure of the adsorbate to be introduced (instead of the equilibrium pressure). The adsorbed amounts (Qads) were determined as the difference between equilibrium pressure before adsorption and after adsorption for each adsorption step.

Liquid Chromatography

The HPLC column of 5 cm long, and 4 mm inner diameter was packed with R-1. Such column was used in High-performance liquid chromatograph Shimadzu equipped with a variable-wavelength ultraviolet detector as well as with a refractive index detector. 2 µL of different butanol solutions (0.08 µg.mL -1 ) were injected onto column. The mobile phase (water:acetonitrile 30:70) was set at a flow rate of 0.1 mL.min -1 . Column temperature was 40°C. Two fractions of the eluted mixture were collected in two vials for further optical identification. The first fraction was taken between 8 and 11 min of retention time, the second fraction was taken between 11 and 14 min of retention time (Fig. 7). As the two chromatographic peaks are not baseline resolved, it can be anticipated that the first fraction contains principally the first eluted enantiomer (R-BuOH) and traces of the second enantiomer (S-BuOH). The opposite situation applies for the second fraction which should mostly contain the S-BuOH enantiomer to come out.

Computational Section

In order to provide microscopic insights of the interactions of the solids with the adsorbed molecules as well as to determine plausible crystal structures, DFT calculations have been performed. First, DFT calculations have probed the impact of the chirality of the butan-2-ol (BuOH) molecules and the phenylethylamine ligand. For that purpose, geometry optimizations on many molecular models were determined with DMol 3 and PW91 GGA density functional and the double numerical basis set containing polarization (DNP) functions were used with high levels of convergence. The results of these simulations are discussed regarding the chirality of the adsorbed molecules (Fig. S14). In order to simplify the system, only the ligand issued from the solid structure has been considered. Second, a plausible crystal structure has been determined using periodic DFT calculations for R-and S-conformation of the ligand. A computational-assisted structure determination has been followed by combining structure determination using XRD refinements and computational geometry optimizations. Again, PW91 GGA density functional and the double numerical basis set containing polarization (DNP) functions were used with high levels of convergence in order to determine the range of unit cell parameters and then to determine the plausible crystal structures. The initial step consists into building the starting configurations in P1 symmetry by bonding the phenylethylamine ligands on the octahedral Ni 2+ centers with a ratio 1:1 with R-or S-conformations. Then geometry optimizations were performed using DFT calculations with free cell parameters. Keep in mind that the co-obtained crystal structures should be dependent from the initial distribution of the ligands. Therefore the simulated unit cell parameters were injected in order to help the refinement of experimental data using Fullprof, as described in the X-ray powder diffraction part. Finally, the last geometry optimization of the unit cell was performed by imposing the experimental parameters. To go further, different organizations of the chiral ligands have been tested in the DFT calculations in order to probe the impact of this organization on the structure energy. The different organizations tested have been summarized in the Fig. 1 and Fig. S6, while the main interactions determined in the structures are reported in the Fig. S7 for the R-and S-conformations as well as for the different distributions of the ligands in the interlayer spaces.

Endly, knowing the crystal structures for the two chiral clathrates, it is therefore possible to estimate the adsorption enthalpy values for R-and S-BuOH and for racemic mixture. For that purpose, Monte Carlo simulations in the NVT ensemble were performed using a home-made code to probe the interactions between the framework of the solid and an enantiopure molecule of alcohol or a racemic mixture (1 for each enantiomer). In this case, it is necessary to calculate both the Van der Waals and the electrostatic interactions. The Van der Waals interactions are determined from the Lennard Jones parameters issued from UFF, 52 with a cut-off for the long interactions fixed at 12.5 Å. Using DFT calculations and the resulting ESP partial charges for the R-and S-butanol molecules (Fig. S15) combined with classical partial charges (issued from the Electronegativity Equalization Method) for the solid framework atoms, it is possible to evaluate the electrostatic interactions using the Ewald summation. For these calculations, a rigid multi-cell structure (4x2x2) was considered and 10 millions of steps were used for both equilibration and production and calculations were performed at 300 K.

Fig. 1 a

 1 Fig. 1 a) Scheme showing the PSM methodology. Colour code: green, Ni; blue, N; red, O; grey, C; b) Crystal structures of R-1 and S-1 obtained by structural optimization (DFT) with an alignment of the ligands.

  These extended architectures consist in octahedral transition metals ions M' 2+ connected through cyano-bridges, provided by square-planar tetracyanometallate complexes [M(CN)4] 2 , generating grid-like layers assembled in 2D structure. The octahedral M' 2+ ions complete their coordination sphere

Scheme 1

 1 Scheme 1 Schematic representation of ligands R-(+)-phenylethylamine and S-(-)-phenylethylamine.

  Ni II-CN-Ni II ) = 2171 cm 1 ) indicating a weak modification of the electronic density. Elemental and Thermo-Gravimetric Analyses (TGA) (Fig. S4) allow the estimation of the ligand and water content giving the following formula [Ni(R-L)1.40(H2O)0.60][Ni(CN)4]•0.3H2O (R-1), [Ni(S-L)1.40(H2O)0.60][Ni(CN)4]•0.3H2O (S-1) and [Ni((+/-)-L)0.94(H2O)1.06][Ni(CN)4]•1.2H2O ((+/)-1

Fig. 2 .

 2 Fig.2. Solid-state (KBr pellet) CD spectra of R-1 (blue), S-1 (red) and (+/-)-1 (green) at room temperature.

Fig. 3 .

 3 Fig. 3. PXRD patterns of 1 (black), R-1 (blue), S-1 (red) and (+/-)-1 (green).

Fig. 4 .

 4 Fig. 4. Adsorption/desorption isotherms of R-BuOH and S-BuOH in the racemic (+/)-1 clathrate.

Fig. 5 .

 5 Fig. 5. R-BuOH adsorption isotherms for R-1 and S-1 solids.

Fig. 6 .

 6 Fig. 6. Adsorption isotherms of R-BuOH, S-BuOH and (+/-)-BuOH with the clathrate R-1.

Fig. 7 .

 7 Fig. 7. Chromatogram of (+/)-BuOH (acetonitrile:water 70:30) using the chiral R-1 clathrate.

1 )Synthesis

 1 The solid 1 (9 mmol, 3.0 g) was activated at 130°C under vacuum for 2 hours. Then, it was cooled to room temperature under argon. The solution of R-(+)-1-Phenylethylamine in chloroform (0.1 M, 180 mL) was added under argon to the activated orange solid. The reaction mixture became gray and was stirred under argon overnight. Then, the precipitate was recovered by filtration, washed with chloroform and dried in the air at 60°C. Lavender solid. IR (KBr, cm -1 ): 2167 ν(NiII -CN-Ni II ); 2125. EDS: 0.49/99.51 (K/Ni). Elemental analysis calcd (%): C, 44.76; H, 4.22; N, 18.55; found (%): C, 44.60; H, 4.09; N, 18.75. Estimated formula for R-1: [Ni(R-(+)-C8H11N)1.4(H2O)0.60][Ni(CN)4Following the same procedure for R-1, compound S-1 was prepared by addition of the solution of S-()-1-Phenylethylamine in chloroform (0.1 M, 180 mL) to the activated solid 1 (9 mmol, 3.0 g) under argon. The reaction mixture became gray and was stirred under argon overnight. Then, the precipitate was recovered by filtration, washed with chloroform and dried in the air at 60°C. Lavender solid. IR (KBr, cm -1 ): 2166, ν(Ni II -CN-Ni II ), 2125. EDS: 0.70/99.30 (K/Ni). Elemental analysis calcd (%): C, 44.73; H, 4.22; N, 18.55; found (%): C, 43.91; H, 3.64; N, 18.42. Estimated formula for S-1: [Ni(S-(-)-C8H11N)1.4(H2O)0.60][Ni(CN)4]•0.3H2O

Table 1 :

 1 Adsorption enthalpy values obtained from Monte Carlo simulations for the different distribution of the ligands in R-1 and S-1 for co-adsorption (in kJ.mol 1 ).

		Adsorption Enthalpies (kJ.mol 1 )
		R-1 (aligned)	R-1 (alternated)
	R-BuOH	62.76	73.86
	S-BuOH	66.94	75.31

  At 25 °C, aqueous solution of K2[Ni(CN)4] (0.8 M, 50 mL) was added dropwise to an aqueous solution of Ni(NO3)2•6H2O (0.8 M, 50 mL). A blue precipitate is formed immediately. After addition, the reaction mixture was refluxed for 1 hour. The precipitate was removing by filtration, washed with ultrapure water several times and dried in the air at 60°C. Light blue powder. IR (KBr, cm -1 ): 2171 (s) ν(Ni II -CN-Ni II ), 2132 (sh).

	EDS: 0.72/99.28 (K/Ni). Elemental
	analysis calcd (%): C, 16.59; H, 2.46; N, 19.69; found (%): C, 16.49; H, 2.09; N, 19.83.

  II -CN-Ni II ); 2125. EDS: 0.36/99.64 (K/Ni).

	Elemental analysis calcd (%): C, 36.40;
	H, 3.93; N, 18.21; found (%): C, 35.24; H, 4.09; N, 17.65. Estimated formula for (+/-)-1: [Ni((+/-)-
	C8H11N)0.94(H2O)1.06][Ni(CN)4]•1.2H2O

Acknowledgements

The authors thank the University of Montpellier, CNRS and PAC of ICGM.

Notes and references