Amal Essaouiba

Teru Okitsu

Rachid Jellali

Marie Shinohara

Mathieu Danoy

Yannick Tauran

Cécile Legallais

Yasuyuki Sakai

Eric Leclerc
email: eleclerc@iis.u-tokyo.ac.jp

Microwell-based pancreas-on-chip model enhances genes expression and functionality of rat islets of Langerhans

Keywords: pancreas, islets of Langerhans, microfluidic biochips, glucose homeostasis, glucagon, insulin

Introduction

The pancreas is a gland organ that plays a key role in endocrine regulation.

The functional units of the endocrine system are the islets of Langerhans. These islets are clusters of cells whose size varies between 20 and 500 μm, with five different cell types: α, β, δ, ε, and γ cells [START_REF] Jouvet | The pancreas: Bandmaster of glucose homeostasis[END_REF][START_REF] Kumar | Pancreas specification: a budding question[END_REF]. The most abundant cells are the glucagon-producing α cells and insulinproducing ß cells. Blood glucose levels are obtained through the interaction of two antagonistic hormones secreted by pancreatic α and β cells. During a period of starvation, when glucose levels are low, glucagon is released by alpha cells to promote glycogenolysis and gluconeogenesis in the liver in coordination with cortisol (a hormone secreted by the adrenal gland). In contrast, insulin secretion from β cells is stimulated by elevated glucose levels and activates glycogenesis in the liver and glucose uptake by muscles and adipose tissues, thereby decreasing postprandial blood sugar levels [START_REF] Baker | Comparison of bioartificial and artificial pancreatic transplantation as promising therapies for Type I Diabetes Mellitus[END_REF][START_REF] Jellali | Chapter 4 -Membrane bioreactors for bio-artificial pancreas[END_REF].

Diabetes mellitus is the most significant endocrine system dysfunction in the pancreas. According to the latest estimates by the International Diabetes Federation (IDF), 1 in 11 adults are living with diabetes in 2019 (463 million people worldwide, IDF official web site, 2019). The incidence of diabetes is increasing dramatically, and is predicted to reach more than 700 million people by 2045 (IDF official web site, 2019). Type 1 diabetes mellitus (T1DM) is an autoimmune disorder which leads to the destruction of insulin-secreting β cells, resuting a lack of insulin production [START_REF] Jellali | Chapter 4 -Membrane bioreactors for bio-artificial pancreas[END_REF][START_REF] Rogal | Stem-cell based organ-on-a-chip models for diabetes research[END_REF]. This pathology affects about 10% of diabetic patients, mostly the young population [START_REF] Aghamaleki | Effect of vitamin D supplementation on pancreatic Β -cell function in patients with Type 1 Diabetes Mellitus and vitamin D deficiency: A clinical trial study[END_REF]IDF official web site, 2019).

The daily insulin administration is currently the most common treatment of T1DM [START_REF] King | Animal models for diabetes: Understanding the pathogenesis and finding new treatments[END_REF]. Type 2 diabetes mellitus (T2DM) is the most prevalent form of diabetes, accounting for 90% of all adult diabetic patients, i.e. 20-79 years old [START_REF] Sardu | Diabetes mellitus and its cardiovascular complications: New insights into an old disease[END_REF]IDF official web site, 2019). T2DM is a metabolic disorder characterized by insulin resistance coupled to impaired insulin secretion from the pancreatic β cells [START_REF] Jun | In vivomimicking microfluidic perfusion culture of pancreatic islet spheroids[END_REF][START_REF] Zbinden | Non-invasive marker-independent high content analysis of a microphysiological human pancreas-on-a-chip model[END_REF]. Insulin resistance is the organs incapacity to respond properly to normal insulin levels (mainly the liver, muscles and adipose tissue, [START_REF] Rogal | Stem-cell based organ-on-a-chip models for diabetes research[END_REF]. The treatment of T2DM involves Lifestyle adjustments and drug therapy such as metformin, sulphonylureas, glitazones and GLP-1 [START_REF] King | Animal models for diabetes: Understanding the pathogenesis and finding new treatments[END_REF][START_REF] Zbinden | Non-invasive marker-independent high content analysis of a microphysiological human pancreas-on-a-chip model[END_REF].

Nowadays, Diabetes is one of the most prevalent chronic diseases [START_REF] Silva | Diagnosis of diabetes mellitus and living with a chronic condition: participatory study[END_REF]. This raises a need for developing pancreatic models to increase knowledge of the underlying mechanisms of diabetes and to screen and identify new anti-diabetic drugs. Pancreatic disease modeling and pertinent models for pancreatic drug screening involve considering the pancreas and its interaction with other organs, such as the liver, muscle, adipose tissues, kideney, [START_REF] Rogal | Stem-cell based organ-on-a-chip models for diabetes research[END_REF] . This is the reason why the disease models involve transgenic and knockout animals [START_REF] King | Animal models for diabetes: Understanding the pathogenesis and finding new treatments[END_REF]. However, animal experimentation is ethically controversial, and often, the animal models developed in other species lose relevance when extrapolating the results to humans [START_REF] Ghaemmaghami | Biomimetic tissues on a chip for drug discovery[END_REF]Merlier et al., 2017;[START_REF] Rogal | Stem-cell based organ-on-a-chip models for diabetes research[END_REF]. Concerning in vitro models, the cell cultures used for drug screening and biomarker discovery are mainly performed in static 2D cultures using conventional Petri dishes or multi-well plates. Although these models have significantly contributed to medical research and drugs screening, they present certain limitations. Today, it seems clear that 2D cultures are poorly representative of human in vivo physiology, metabolism and toxicity, due to the physiological gap between the cells cultivated in static 2D mode and human cells as they exist in their native state (3D, dynamic mode) and the lack of physiological integration between cells and organs (Merlier et al., 2017). As a result, it is essential to improve these basic plate cultures to understand and model metabolic processes and human disorders. This is why many groups are developing tissue-engineering and 3D culture processes in order to provide a more appropriate micro-environment for tissue maintenance and development. This environment must reproduce, as closely as possible, the characteristics found in vivo.

The organ-on-chip approach is one way of mimicking organ physiology to help in the development of therapeutic solutions and pharmacological studies [START_REF] Huh | Microengineered physiological biomimicry: organs-on-chips[END_REF]Bathia and Ingber 2014). This approach has many advantages to reproduce the characteristics of physiological microenvironments, including three-dimensional architectures, cell-cell interactions and dynamic flow that ensures the transport and exchange of culture medium, hormones, metabolic waste and other chemicals (merlier et al., 2017;[START_REF] Lee | Microphysiological analysis platform of pancreatic islet β-cell spheroids[END_REF]Zbendin et al., 2020). Unlike the static cultures in Petri dishes, organ-on-chip approch provides the possibility to perform co-cultures, where the different cell types can be cultivated in separated microbioreactors and the cell-cell interactions are ensured by soluble factors exchange (Merlier et al., 2107).

The co-culture of two or more organ is a promising tool to study multi-organs diseases such as T2DM [START_REF] Rogal | Stem-cell based organ-on-a-chip models for diabetes research[END_REF]. In the last years, organ-on-chip technology has been used to reproduce pancreas in vitro models [START_REF] Schulze | A 3D microfluidic perfusion system made from glass for multiparametric analysis of stimulus-secretion coupling in pancreatic islets[END_REF][START_REF] Li | 3D-templated, fully automated microfluidic input/output multiplexer for endocrine tissue culture and secretion sampling[END_REF][START_REF] Brooks | Macro-tomicro interfacing to microfluidic channels using 3D-printed templates: application to time-resolved secretion sampling of endocrine tissue[END_REF][START_REF] Lee | Microphysiological analysis platform of pancreatic islet β-cell spheroids[END_REF]2018;[START_REF] Zbinden | Non-invasive marker-independent high content analysis of a microphysiological human pancreas-on-a-chip model[END_REF]. A literature review highlighted that most of the current microfluidic platforms have been designed mainly for islet quality assessment for subsequent in vivo implantation [START_REF] Rogal | Stem-cell based organ-on-a-chip models for diabetes research[END_REF]. In parallel, some recent works have also demonstrated the potential of organ-on-chip technology for more complex pancreatic physiopathology analysis (Bauer et al., 2017;[START_REF] Rogal | Stem-cell based organ-on-a-chip models for diabetes research[END_REF].

In this work, we developed a simple microfluidic biochip for assessment of pancreatic islet function and long-term cultures. We also presented and demonstrated that the islets functionality was maintained in microfluidic biochips when compared to conventional islet cultures inside polystyrene (TCPS) dishes.

Material and methods

Device design and fabrication

The microfluidic biochip consists of a cell culture chamber manufactured with two polydimethylsiloxane (PDMS) layers (Fig. 1A). The micro-structured bottom layer, used for trapping the islets, is composed of 600 micro-wells of 400 µm in diameter (depth of 300 µm), and spaced 50 µm apart (Fig. 1B). The second PDMS layer, with a reservoir (depth of 100 µm), is placed on top of the first layer and includes an inlet and outlet for culture medium perfusion. A micro-channel network placed at the inlet and outlet of each layer ensures homogeneous culture medium distribution within the biochip (Fig. 1A).

The PDMS biochip was manufactured using the replica molding process. First, photolithography was performed to create the mold masters of the bottom and top layer of the biochips using SU-8 photosensitive resin. Then, the PDMS prepolymer (in a mixture of 10:1 base polymer: curing agent; Sylgard 184, Dow Corning) was poured on to the SU-8 master and cured for 2 h at 75°C. The surfaces of the PDMS layers obtained were activated with reactive air plasma (1 min; Harrick Scientific) and brought together immediately to form an irreversible seal.

Pancreatic islets isolation

Islets of Langerhans were isolated from male Wistar rats (8-9 weeks old, 200-300 g) (CLEA Japan, Inc, Tokyo, Japan) following a slight modification to the protocol described by [START_REF] Yonekawa | A new mouse model for intraportal islet transplantation with limited hepatic lobe as a graft site[END_REF] and [START_REF] Kiba | High-quality RNA extraction from rat pancreatic islet[END_REF]. All animal experimentation procedures were carried out in accordance with the guidelines of the University of Tokyo and the Japanese Ministry of Education.

The rats were anesthetized with isoflurane inhalation solution (Pfizer). After clamping of all irrigation blood vessels, the enzymatic solution (Liberase™ TL by Roche) was injected through the bile duct, previously identified and clamped. After the pancreatectomy, there was selective chemical digestion of the organ at 37°C for 30 min with Liberase TL/ ET-K solution (ET-Kyoto solution, Otsuka Pharmaceutical).

The digestion was followed by washing and purification steps using a discontinuous OptiPrep® (Sigma-Aldrich) density gradient. The islets of Langerhans were then identified, individually hand-picked with a Pasteur pipette under a stereomicroscope (Leica S9 D), and transferred to a cold preservation solution made of UW solution (University of Wisconsin, [START_REF] Kneteman | Islet isolation from human pancreas stored in UW solution for 6 to 26 hours[END_REF] complemented with Miraclid (Mochika pharmacy, Japan) and heparin (Mochika pharmacy, Japan). After assessing and counting the islets, the tissue was stored at 4 °C until starting the culture in order to maintain full functional properties as shown in [START_REF] Kimura | Improved hypothermic short-term storage of isolated mouse islets by adding serum to preservation solutions[END_REF].

Pancreas on chip and Petri culture

The biochips and perfusion circuits (silicone/Teflon tubing and bubble trap) were sterilized by autoclaving and dried in an oven. The biochips were then assembled with the perfusion system and filled with culture medium in order to remove air bubbles and moisturize the circuits. The bubble trap was used as a reservoir interconnected to the biochips by the silicone/Teflon tubing of 0.65 mm in diameter. The preconditioning process was carried out for one hour at 37° C in the incubator. The entire setup is presented in Fig. S1 (supplementary file).

The pancreatic islets in the preservation solution were washed with cold culture medium and gently diluted in the appropriate amounts in order to ensure fair and even distribution of the tissue in the biochips and Petri culture. The estimated number of islets per biochip or well is ≈40. In order to minimize damage to the islets, wide orifice pipette tips with low binding were used throughout the handling process.

Once the islets were loaded in the biochips from the inlet port or seeded in the 24well plate, the counting step took place under the microscope in order to keep a record of the islets per biochip and/or well. The cultures were continuously maintained at 37 °C in a 5% CO 2 supplied incubator.

Two groups of study (biochip and Petri) and 4 conditions were established:

Petri control; Petri with GLP-1; biochip control and biochip with GLP-1. The basal culture medium used in our study were the classic RPMI 1640 Medium (Gibco, 2.5 mM of glucose) supplemented with 10% FBS (Gibco), 100 units/mL of penicillin, 100 mg/mL streptomycin (Gibco) and GlutaMAX™ (Gibco™) at 10 mM. For the hormone-stimulated media, GLP-1 (Peprotech, USA) was added for a final concentration of 100 nM. The biochips were perfused in a circuit loop containing ≈2 ml of culture medium with a peristaltic pump (flow rate of 20 µL/min). The medium was renewed every 2 days. In static conditions, the islets were seeded in 1 mL of medium/well and the culture medium was exchanged every day.

Islets viability

At the end of the experiment, the islets were incubated in a solution of propidium iodide (PI) at 4.5 µmol/L and calcein-AM at 2 µmol/L (Cellstain kit, Dojingo) in RPMI 1640 medium for 30 min in the dark. Then, the samples were washed with RPMI 1640 medium and observed under an epifluorescence microscope (Olympus, Japan). The size of necrotic core was quantified by ImageJ software (NIH, Bethesda, Maryland) using the collected images. The area of the cells stained with PI was measured and normalized by the islet area.

RTqPCR assays

Total RNAs were extracted and purified from samples using a hybrid protocol that combines Trizol™ Reagent (Life Technologies) and RNeasy Mini Kit (QIAGEN 74104) following the manufacturer's instructions. The concentrations and qualities of the RNAs extracted were assessed using a BioSpec-nano (Shimadzu Scientific Instruments). Reverse-transcription into cDNA was performed from 0.5 μg of total RNA using the ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO).

Real-time quantitative PCR was then performed with the THUNDERBIRD SYBR qPCR Mix (TOYOBO) according to the manufacturer's protocol and a StepOnePlus Real-Time PCR system (Applied Biosystems). The primer sequences of the genes are shown in Table S1. β-Actin was used as the reference gene and fresh isolated islets (days 0) as the reference sample for the normalization of gene expression data.

Immunostaining

Immunofluorescent detection was investigated at the end of the experiment (day 5). We selected to stain the GCK because the coding protein glucokinase is a key enzyme using glucose as substrate. We also stained two important markers in the pancreatic islets: insulin and glucagon that are markers of -cells and -cells, respectively.

After transfer to an untreated TCPS 24-well plate (not a plate treated for cell cultures, to prevent the islet cells from attaching to the plate), the islets were washed with phosphate buffer saline solution (PBS) and fixed in paraformaldehyde 4% at 4°C for 24 hours. In order to perform the immunostaining in a 3D structure, the islets were permeabilized with 1% Triton X100 in PBS for 3 hours at 4°C and washed 3 times with PBS for 30 min. Then, the islets were blocked with a gelatin buffer for 24 hours at 4°C. Primary antibodies (anti-insulin [ab6995, Abcam], anti-glucagon [ab167078, Abcam] and anti-glucokinase [sc-17819, Santa Cruz]) were incubated for 48 hours at 4°C in a BSA/PBS solution. Secondary antibodies coupled with Alexa Fluor fluorochromes (Glucagon: anti-rabbit Alexa Fluor 680 [A-21109, ThermoFisher],

Insulin: anti-mouse Alexa Fluor 594 [ab150116, Abcam] and Glucokinase GCK: antimouse Alexa Fluor 647 [ab150107, Abcam]) were further incubated overnight in a BSA/PBS solution at 4°C in the dark. Finally, the nuclei were stained with DAPI (342-07431, Dojindo) at 1/1000 for 30 min at room temperature (RT) in the dark. All the incubations and washing steps were carried out using a shaker. Observations were made with an Olympus IX-81 confocal laser-scanning microscope.

Functional assays

Glucose-stimulated insulin secretion (GSIS) assays

After 4 days of culture, we performed a low high glucose stimulation. In biochips, the culture medium was removed from the bubble trap and the circuit (biochip, tubing and bubble trap) was washed with a 0-glucose solution (D-MEM, No Glucose, Wako) for 2 hours. This washing 0-glucose solution was removed from the bubble trap, and 1 mL of fresh 0-glucose was added and perfused for 2 hours. After this low glucose perfusion, the islets were exposed to a high glucose culture medium for 2 hours (D-MEM high Glucose, Wako, 25 mM of glucose). For that purpose, the low glucose solution was removed from the bubble trap and replaced with 1 mL of high glucose. In a Petri dish, this protocol led to 2h of 0-glucose exposure, followed by another 2h of 0-glucose exposure and finally 2h of high glucose stimulation. At the end of the assays, basal media were re-established for all conditions.

GLP-1 stimulations

In order to test the response of the islets to drug stimulations, we exposed the cultures to GLP-1. For that purpose, we followed the same protocols described in section 2.3, to which we added 100 nM of GLP-1 at each culture medium change step.

Insulin, glucagon and C-peptide measurements

The hormones released into the culture medium were assessed using ELISA assays, following the manufacturer's protocol. To measure each hormone concentration, we used a rat Insulin ELISA kit (10-1250-01; Mercodia) for insulin, a Glucagon DuoSet ELISA kit (DY1249; R&D Systems) with the DuoSet ELISA Ancillary Reagent Kit 2 (DY008; R&D Systems) for glucagon, and a rat C-Peptide ELISA kit (10-1172-01; Mercodia) for C-peptide.

Glucose and lactate quantification

Glucose and lactate were measured using an YSI 2950 Biochemistry Analyzer.

For that purpose, 150 µL of culture medium were inserted into the analyzer.

Measurements were based on a direct reading of L-lactate (L-lactic acid) and glucose in the culture medium by the YSI enzyme sensors, as the enzymes L-lactate oxidase, and glucose oxidase are respectively immobilized in the lactate and glucose sensors.

Statistics

The rat islets were used in 6 to 12 biochips according to the experiments. The RTqPCR were performed on three samples for each condition in three independent experiments (n=3x3). The measurements of glucose, lactate, insulin, C-peptide and glucagon were also performed in triplicate. The Kruskal Wallis test was performed for the statistical analysis.

Results

Characterization of islet biochip cultures when compared to Petri dish cultures

Viability assay showed highly viable islets in the biochip

After extraction (Fig. 2A), the islets were cultivated in Petri dishes (Fig. 2B) and in biochip dynamic culture for 5 days (Fig. 2C). In the device, the islets sedimented into the microwells located at the bottom of the culture chamber. Then, we confirmed that the islets had not been washed away by the flow rate after 5 days of perfusion. Indeed, the number of islets seeded at the beginning of the experiments and collected at the end of the perfusion remained similar, as shown in Fig. 2D. In addition, the islets presented a round shape at the end of the perfusion (Fig. 2B,2C and Fig. S2, supplementary file). The typical size of the islets was about 150  50 microns.

The fluorescent images of islets stained with calcein AM/PI are presented in Fig. 2E. We found a different size to the necrotic core of the islets between static and dynamic conditions after 5 days of culture. Nevertheless, the viability was higher in the islets cultivated in the biochips inasmuch as the IP staining was weaker when compared to Petri situations. The quantification of the necrotic core showed that the proportion of dead cells in dynamic biochip was significantly lower when compared to the Petri culture. The normalized size of necrotic core was of 0.34±0.15 and 0.12±0.03 for islets cultivated in Petri and biochip, respectively (Fig. S3, supplementary file).

RTqPCR analysis revealed higher mRNA levels of pancreatic islets markers in biochip cultures

At the end of the experiments, we compared the mRNA levels of the cells in the islets cultivated in biochips and Petri dishes (Fig. 3). The markers related to maintaining islet differentiation, such as Reg3a and Neurod, were upregulated in the biochips when compared to the Petri cultures (fold change, FC, of 190 and 13, respectively). In addition, the markers related to islet functions such as App (4.3 FC), Ins1 (1.7 FC), Sst (2.8 FC) and Gcg (2.1 FC) were also upregulated in the biochips.

Finally, the levels of several receptors and transporters such as Gcgr, Insr, and Glut2 were 3.2 to 5.5 times higher in the biochip compared to the Petri dishes (Fig. 3).

Immunostaining confirmed the expression of pancreatic islets markers and glucose regulators in both biochips and Petri dishes

The immunostaining of the islets, prior to inoculation in the biochips, demonstrated that the islets were positive for insulin and glucagon (day 0, Fig. 4A and Fig. S4 in supplementary file). At the end of the experiments, the islets from the Petri dishes and biochips were positive for insulin and glucagon as shown in Fig. 4A and Fig. S4 (supplementary file). We observed three types of cell populations, insulin positive cells, glucagon positive cells and a third subpopulation expressing both insulin and glucagon (those bihormonal cells may reflect a partial switch from cells to -cells, supplementary file). We confirmed also that the islets in both culture modes were positive for GCK (Fig. 4B and Fig. S5 in supplementary file), consistently with the composition of the culture medium containing 2.5 mM of basal glucose (nb: GCK, in the pancreas, plays a role in glucose-stimulated insulin secretion).

Functional assays revealed higher insulin secretion in the islets in the biochips

The basal functionality of the islets was demonstrated by measuring insulin, Cpeptide and glucagon secretion. At the end of each experiment, the islets were counted to normalize the data. We found that the insulin concentration in the culture medium decreased in both biochips and Petri dishes. However, the biochip cultures contributed to maintaining relatively high levels for all experiments until day 4, whereas a drop occur between day 3 and day 4 in Petri (Fig 5A).. Biochip cultures contributed to maintaining insulin secretion close to 130 ng/islet/day until day 4, and decreasing to 44 ng/islet/day at day 5. In the Petri dishes, we found a significant reduction in days 3 and 4, leading to measured secretion close to 30 ng/islet/day before dropping to 17 ng/islet/day at day 5. We also observed higher secretion of Cpeptide in the biochips when compared to the Petri dish cultures, as shown in Fig 5B.

Secretion was about 5 times higher in the biochip from days 3 to 5. C-peptide secretion in Petri dish decreased significantly since day 2, whereas it decreased only at day 5 in the biochips.

The glucagon levels were higher in the biochips than in the Petri dishes, as shown in Fig. 5C. Furthermore, the levels remained constant in the biochip cultures for 4 days of culture, close to 1000 pg/islet/day, before decreasing to 500 pg/islet/day on day 5. In the Petri dishes, glucagon secretion decreased from day 3 to day 4 and leading to a drop from 560 to 180 pg/islet/day between day 2 to day 5. The ratio between insulin and glucagon secretion is shown in Fig 5D . In the biochips, the ratio increased from 130 to 250 between days 2 and 4, before dropping to 85 on day 5. In the Petri dishes, the ratio decreased continuously from 380 to 80, but with high intra and extra experiment variability.

Finally, glucose and lactate levels were measured during culture. As the culture medium was frequently changed during the experiments, we observed that glucose levels remained relatively stable and very high in our experiments. Both in the Petri dishes and biochips, we were unable to detect any significant glucose depletion (Fig 5E). Furthermore, lactate production was higher in the biochips than in the Petri dishes (Fig 5E).

Pancreatic islet response to stimulations demonstrated functional and active biochip culture conditions

Low-high glucose stimulations

After 4 days of culture, we performed a low-high glucose stimulation. The islets demonstrated lower insulin secretion in low glucose stimulation when compared to high glucose stimulation. To evaluate islets responsiveness to low/high glucose stimulation, we calculated the GSIS (glucose-stimulated insulin secretion) index by dividing insulin measured in high-glucose and low-glucose media (Fig. 5F).

The GSIS index values were of 5 and 2.1 in the Petri dishes and biochip, respectively.

These results indicate that glucose response of islets was maintained in both culture modes. However, we should mention that we observed wide variability in this assay inasmuch as some Petri dishes or biochips were not induced, thus leading to a significant error bar and dispersion.

Effect of glucagon-like peptide-1, GLP-1, drug

The GLP-1 stimulations contributed to the modification of mRNA levels, both in Petri dishes and biochips (Fig 6A). GLP-1 increased the mRNA levels of the receptors Glp-1r, Gcgr, and of the Reg3a, Ins1, Ins2 and Stt genes in the biochips (when compared to non-treated biochip controls) and downregulated Neurod. In well plates, we also found upregulation of Gcgr, Insr, Ins2, and downregulation of Neurod (Fig. S6, supplementary file). Furthermore, the mRNA levels in the GLP-1-treated biochips were upregulated when compared to GLP-1-treated Petri dishes (data not shown). Fig. 6B shows the insulin immunostaining of islets treated by GLP-1. The data demonstrated a high fluorescence intensity in GLP-1-treated Petri and biochip. We also found that the levels of the GCK protein in the islets were not over-expressed by the GLP-1 treatment (Fig. 6B). At the functional levels, insulin secretion was increased in both Petri dishes and biochips by the GLP-1 treatments (Fig 7A). On days 2, 3 and 4, the levels of insulin in the biochips were up to 3 times higher than those attained in control. However, after 5 days, this induction was weaker (2 times).

We then performed the low-high glucose stimulation tests after pre-treatment with GLP-1. As expected, the drug did not inhibit the effect of the high / low glucose stimulations and maintained a high level of insulin production in the biochips under high glucose stimulation (Fig 7B). In this assay, the low / high glucose stimulation led to an insulin over production of 2.2 and 4 in biochips and Petri, respectively (GSIS index of 2.24 and 4, respectively). Furthermore, we did not observe variability in the assay when compared to the untreated conditions (cf. Fig 5F).

Discussion

In this study, we investigated the performance of islets of Langerhans in a closed loop microfluidic system. Several investigations have reported microfluidic techniques for keeping islet or spheroid cultures on chip, including the pancreas [START_REF] Tan | A trap-and-release integrated microfluidic system for dynamic microarray applications[END_REF][START_REF] Rogal | Stem-cell based organ-on-a-chip models for diabetes research[END_REF][START_REF] Lee | Microphysiological analysis platform of pancreatic islet β-cell spheroids[END_REF][START_REF] Mohammed | Microfluidic device for multimodal characterization of pancreatic islets[END_REF].

Those devices are based on a flow trapping process [START_REF] Tan | A trap-and-release integrated microfluidic system for dynamic microarray applications[END_REF][START_REF] Rogal | Stem-cell based organ-on-a-chip models for diabetes research[END_REF]. We built a simple culture biochip where the pancreatic islets can be trapped by sedimentation in the bottom microstructure. This type of trapping was also reported by [START_REF] Jun | In vivomimicking microfluidic perfusion culture of pancreatic islet spheroids[END_REF] In our biochip, the microstructure contributed to create an array of 600 microwells allowing to trap an important quantity of islets.

The depth of microwells made it possible to protect the islets from flow circulation and thus prevent them from being washed away and from mechanical damage. As a result, we maintained a constant number of islets between the beginning and the end of the perfusion. Furthermore, due to sedimentation in the microwells, the islets were protected from the fluid shear stresses created by the laminar flow.

In biochip, we observed higher viability for the cells located in the center of the pancreatic islets when compared to the Petri dish cultures. This was illustrated by the viability assay in which a larger number of dead cells was observed in the center of the islets when they were cultivated in Petri dishes. Central necrosis and apoptosis in pancreas islets are due mainly to the high density of the tissue and the lake irrigation as has been widely reported in literature [START_REF] Giuliani | Central necrosis in isolated hypoxic human pancreatic islets: evidence for postisolation ischemia[END_REF][START_REF] Moritz | Apoptosis in hypoxic human pancreatic islets correlates with HIF-1alpha expression[END_REF].

Several hypotheses are proposed to explain central necrosis and apoptosis in pancreatic islets (and also in cell spheroids), including deprivation of oxygen, nutrients and serum as a result of limited diffusion [START_REF] Giuliani | Central necrosis in isolated hypoxic human pancreatic islets: evidence for postisolation ischemia[END_REF]. In the case of the pancreatic islets, the blood micro-vessels usually reach the center of the structure in vivo but the microvasculature gets closed in ex vivo tissue (Jansson et al. 2016). In parallel, microfluidic cultures have been shown to improve the viability of spheroid tissue by reducing the central necrotic core thanks to control of local glucose concentrations and local oxygenation through the dissolved oxygen provided by the medium flows [START_REF] Barisam | Prediction of necrotic core and hypoxic zone of multicellular spheroids in a microbioreactor with a U-shaped barrier[END_REF][START_REF] Baye | Microfluidic device flow field characterization around tumor spheroids with tunable necrosis produced in an optimized off-chip process[END_REF]. As a result, those literature reports appeared consistent with our findings.

In our investigation, we found that the microfluidic culture maintained and improved pancreatic regeneration and maturation markers of the rat islet cells. This was illustrated by higher levels of mRNA in important pancreatic islet markers, including receptor genes (Gcr and Insr), hormone secretion-related genes (App, Sst and Ins1) and differentiation genes (Neurod and Reg3a). More particularly, we found a 190-fold upregulation of Reg3a, which is an islet regeneration marker [START_REF] Coffey | Microarray analysis of rat pancreas reveals altered expression of Alox15 and regenerating islet-derived genes in response to iron deficiency and overload[END_REF] involved in the pro-islet gene cascade and their protection against induced diabetes mellitus [START_REF] Xiong | Pancreatic islet-specific overexpression of Reg3beta protein induced the expression of pro-islet genes and protected the mice against streptozotocininduced diabetes mellitus[END_REF]. In addition, Neurod was 13 times higher in the biochips compared to Petri dish levels. Neurod is an important gene (an insulin trans activator) required to maintain functional maturity in pancreatic beta cells, including insulin production through Ins1 [START_REF] Gu | Pancreatic β cells require NeuroD to achieve and maintain functional maturity[END_REF]. We also consistently found upregulation of the Ins1 gene in the biochips, which is consistent with Ins1 silencing in Neurod KO mice [START_REF] Gu | Pancreatic β cells require NeuroD to achieve and maintain functional maturity[END_REF]. Neurod KO-mice expressed the Ins2 gene and were thus able to produce insulin in a glucose stimulation test, which also appeared consistent with our findings in which Ins2 was commonly expressed in both the Petri dishes and the biochips [START_REF] Gu | Pancreatic β cells require NeuroD to achieve and maintain functional maturity[END_REF].

In parallel, an over-expression of the insulin receptor Insr was observed. Insr is involved in controlling glucose-stimulated insulin secretion via its relationship with insulin gene expression, Glut2 and Pdx1 [START_REF] Wang | Knocking down insulin receptor in pancreatic Beta cell lines with lentiviral-small hairpin RNA reduces glucose-stimulated insulin secretion via decreasing the gene expression of insulin, GLUT2 and Pdx1[END_REF]. We consistently observed a tendency for higher mRNA levels in the Glut2 and Pdx1 genes in the biochips. Upregulation of Gcgr, the glucagon receptor, in the biochips also indicated a probable beneficial effect of the microfluidic cultures on the alpha cells inasmuch as the lack of glucagon receptors is correlated with several pancreas disorders and alteration to glucose homeostasis, such as hyperplasia and hyperglucagonemia [START_REF] Charron | Lack of glucagon receptor signaling and its implications beyond glucose homeostasis[END_REF].

At the functional level, we analyzed the performances of the pancreas islets when they were cultivated in the biochips. At first, this was illustrated by the kinetics of the secretion of insulin in the culture medium. Although the overall secretion decreased between the first day and fifth day of culture in both culture conditions, the biochip levels remained higher when compared to those of the Petri dishes. Secondly, the high/low glucose stimulation assay demonstrated that islet response to stimuli was preserved in our biochip culture. However, in the biochips, the protocol required an extended washing process to remove the insulin and remaining glucose from the basal culture medium in the perfusion circuit (mainly due to the dilution of high glucose solution in washing solution that would lead to bias stimulation). This need of washing process in the biochips, when compared to conventional Petri dishes, has already been reported in pancreas-on-chip investigations [START_REF] Rogal | Stem-cell based organ-on-a-chip models for diabetes research[END_REF].

Nevertheless, enhancements of basal islets functions such as insulin secretion and glucose-induced insulin secretion in microfluidic devices have been observed consistently in the literature [START_REF] Sankar | Culturing pancreatic islets in microfluidic flow enhances morphology of the associated endothelial cells[END_REF]. In our dataset, this over-secretion of insulin was not correlated with over-expression of Gck, as demonstrated by the immunostaining. The glucose-stimulated insulin secretion was regulated by the rate of glucose metabolism within β cells, and a key event in this process is the phosphorylation of glucose by glucokinase (the coding gene is Gck, [START_REF] Wu | Oxidative stress is a mediator of glucose toxicity in insulin-secreting pancreatic islet cell lines[END_REF]). Although we worked in a closed loop perfusion circuit, the flow rate led to continuously renewing the culture medium locally in the biochips. Furthermore, the level of glucose remained high in the medium, probably due to the number of islets.

The microfluidic culture completely modulated the chemical cellular microenvironment (via a complex gradient of molecules, a balance between diffusion and convection, cellular consumption) leading to complex signals [START_REF] Young | Fundamentals of microfluidic cell culture in controlled microenvironments[END_REF], Halldorson et al., 2015). In parallel, the microwells almost certainly influenced the local nutrient islets' microenvironment (a balance between glucose consumption, glucose renewal, secreted insulin concentration, etc…). As a result, additional local measurements (via integrated sensors) and numerical simulations are needed to understand the complex kinetics of insulin secretion in relation to local glucose concentrations in our biochips.

Finally, we tested the effect of the glucagon-like peptide-1 (GLP-1) drug in both culture conditions. GLP-1 physiologically induces glucose-dependent insulin secretion from β-cells and GLP-1 analogues improve hyperglycemia in T2D patients [START_REF] Drucker | The incretin system: glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors in type 2 diabetes[END_REF][START_REF] Defronzo | Effects of exenatide (exendin-4) on glycemic control and weight over 30 weeks in metformin-treated patients with type 2 diabetes[END_REF], Vilsbol et al., 2007). Furthermore, GLP-1 treatment increases Gck activity [START_REF] Ding | Glucagon-like peptide 1 stimulates post-translational activation of glucokinase in pancreatic cells[END_REF]. However, long term GLP-1 exposure (about 18h) also resulted in the promotion of the metabolic reprogramming of β-cells through mTOR-dependent HIF signaling, and independently of Gck posttranslational activation [START_REF] Carlessi | GLP-1 receptor signalling promotes β-cell glucose metabolism via mTORdependent HIF-1α activation[END_REF]. In the present study, GLP-1 stimulation clearly upregulated several pancreatic islet genes such as Glp-1r and Gcgr, in both Petri dishes and biochips. In addition, those mRNA levels were upregulated in the biochips compared to the Petri dishes when the GLP-1 was loaded. At the functional level, GLP-1 induced an increase in insulin secretion in both culture modes (as confirmed by the immunostaining of insulin and the insulin extracellular concentrations in the medium). The secretion levels in the culture medium were found to be higher in the biochips. However, we did not find any of the inhibitory effect we expected in the alpha cells of the islets of Langerhans (glucagon secretion) (Tudori et al. 2016). In our biochips, we found mRNA Glut2 over-expression compared to the Petri dishes, leading to the suspicion of higher glucose transport, and thus metabolism, in the islets. We did not find an effect of GLP-1 on the Gck at the immunostaining fluorescent level. Although these findings seems consistent with the observations made by [START_REF] Carlessi | GLP-1 receptor signalling promotes β-cell glucose metabolism via mTORdependent HIF-1α activation[END_REF], more extensive investigations are needed to confirm the underlying mechanism in biochips.

Conclusion

In this work, we studied the behavior of rat islets of Langherans when cultivated in microfluidic biochips or in Petri dishes. The microfluidic biochips cultures maintained high islet viability throughout the 5 days of culture. More particularly, several important pancreatic islet genes, including Reg2a, Neurod, Insr, Gcgr, Glut2, Ins1, App and Stt were overexpressed in biochips cultures (compared to islets cultivated in Petri). The islets were able to secrete insulin and glucagon, as well as to respond to GLP-1 drug stimulation and high-low glucose test. Furthermore, the levels of insulin secretion appeared higher in biochips when compared to the Petri dishes. Our dataset illustrated the fact that the microfluidic culture is beneficial for maintaining in vitro maturation and functionality of islets of Langerhans. We believe that our results are encouraging for the development of functional pancreas in vitro models using the advantages of organ-on-chip technology.

Acknowledgments

A part of the project was funded by the French National Research Agency (ANR-16-RHUS-0005). Amal Essaouiba's PhD grant was supported by the French Ministry of Research and Higher Education. We thank the CNRS/IIS UMI 2820, the UMR 7338 and the universite de technologie de Compiegne for their mobility supports to A. Essaouiba to Tokyo.


Dr Alexey N Bilyachenko
email: bilyachenko@ineos.ac.ru

Prof. V Alena N N Kulakova

Prof Mikhail M A A Levitsky

Alexander Korlyukov

Victor N Khrustalev

Anna Vologzhanina

Aleksei A Titov

Pavel V Dorovatovskii

Lidia S Shul'pina

Frédéric Lamaty
email: frederic.lamaty@umontpellier.fr

N Bilyachenko

Alexander A Korlyukov

Anna V Vologzhanina

Frdric Lamaty

Bantreil

Benot Villemejeanne

Prof Cindy J Ruiz

Jean Martinez

Elena S B Shubina

Shul'pin
email: shulpin@chph.ras.ru

Dr A V Korlyukov

Dr A A Vologzhanina

L S Titov

Prof. E S Shul'pina

A N Shubina

Nesmeyanov

Dr A A Khrustalev

Titov

Prof A A Korlyukov Pirogov

Dr F Lamaty

Dr X Bantreil

Martinez

Prof G B Shul'pin Semenov

Prof G B Shul'pin Plekhanov

Ionic Complexes of Tetra-and Nonanuclear Cage Copper(II) Phenylsilsesquioxanes: Synthesis and High Activity in Oxidative Catalysis

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Introduction

Among the huge variety of 3 D polycyclic (cage) metal-containing complexes, [1] those based on structural silsesquioxane (RSiO 1.5 ) fragments [2] attract special attention as systems that simulate metal-center-grafting to silica surfaces [3] perfectly in heterogeneous catalytic applications. [4] Such compounds (cage-like metallasilsesquioxanes, CLMSs) are also (i) active in homogeneous catalysis, [5] (ii) demonstrate specific (spin glass) magnetic properties, [6] or (iii) provide opportunities for unusual reactivity [7] and structural reorganization governed by thermal treatment. [5a, 8] The most demanded synthetic approaches to CLMSs include reactions using cubane, [9] cyclic, [10] and trisilanol [2,11] synthons as well as self-assembly [11b, 12] or transmetalation [11b, 13] procedures. Here we report the preparation of new types of CLMSs following the "double ligation principle" of self-assembly. This new synthesis method implies the use of two types or reactants that are able to ligate metal ions in the process of cage assembly: an O-ligand (silsesquioxane) and a P-ligand (1,2-bis(diphenylphosphino)ethane; dppe). It is important to differentiate this approach from several syntheses of CLMSs that involve bidentate phosphine, in which phosphine served as a component of the initial metal complex. [14] Here we show the possibility of a double (O, P) ligation approach to CLMSs, as well as the tendencies of the supramolecular aggregation of products and their catalytic properties in the oxidation of CH compounds and formation of amides.

Herein, we describe an approach to cage metallasilsesquioxanes by self-assembly with 1,2-bis(diphenylphosphino)ethane as a key reactant. This approach allowed us to achieve a unique family of complexes that includes anionic tetra-and nonanuclear cage copper(II) sodium silsesquioxane and cationic copper(I) 1,2-bis(diphenylphosphino)ethane components. Additional representatives of this intriguing metallasilsesquioxane family (Cu 9 Na 6 and Cu 9 Na 3 Cs 3 ) were obtained through the replacement of the original ethanol-based reaction medium by DMSO. The fascinating structural peculiarities of all products were established by using XRD and topological studies. Initial tests for the application of the synthesized complexes as catalysts revealed their very high activity in the homogeneous oxidation of alkanes and alcohols to produce alkyl hydroperoxides, ketones, and amides.

Results and Discussion

Synthesis

To date, several instances of the use of phenyltrialkoxysilanes as simple but efficient educts in the self-assembly of CLMSs have been reported. [11b, 12-13] However, no examples of the involvement of the bidentate P-based ligand were among them. As we are interested in the principles of double-ligated (silsesquioxane/phosphine) CLMSs formation, we have performed several parallel reactions of the synthesis of Cu-containing CLMSs. A three-component mixture was used for this self-assembly reaction: (i) highly reactive phenylsiloxanolate [(PhSiO 1.5 )(NaO 0.5 )] n moieties (prepared in situ from PhSi(OEt) 3 and NaOH) as the silsesquioxane component, (ii) 1,2-bis(diphenylphosphino)ethane as the phosphine component, and (iii) CuCl 2 as a source of transition metal ions. Among the combinations of solvents used for the synthesis (DMF, acetonitrile, THF, 1,4-dioxane, etc.), only ethanol was able to assist the assembly of the target complex (Scheme 1).

Two types of compounds could be isolated this way, depending on the NaOH loading (1 and 2; Figure 1). Com-Scheme 1. Solvent variation during the synthesis of Cu,Na-phenylsilsesquioxanes. The use of EtOH provided 1 (42 %) and 2 (17 %). pounds 1 and 2 contain a CLMS fragment as an anionic component, and Cu(dppe) 2 fragments serve as cationic fragments to balance the charge.

This intriguing feature of CLMS formation, to the best of our knowledge, has not been described previously in the chemistry of metallasilsesquioxanes. Several unusual features of both complexes are notable. Previously, the presence of additional organic ligands in the CLMS formation medium provoked the formation of complexes in which both silsesquioxane and additional ligands (e.g., triphenylphosphine oxide, [15] N,N,N',N'-tetramethyl-p-phenylenediamine, [16] trimethylphosphine, [17] tris [2-(dimethylamino)ethyl]amine, [18] pyrazine [19] ) coordinate metal ions of the same cage compound.

As opposed to this "ligand synergy" situation, 1 and 2 represent another "ligand antagonism" principle of complexation. Namely, the Cu ions of both 1 and 2 are redistributed between different (O and P) ligands to give rise to the two-component products, in which silsesquioxane O-ligands form an anionic cage fragments and phosphine P-ligands provide cationic Cu(dppe) 2 fragments. Notably, the compositions of the silsesquioxane cage components of 1 and 2 are different to each other. At first sight, the cage component in 1 is well known in the chemistry of CLMSs. [11b, 12-13] Its sandwich-like structure is characterized by the presence of the two cyclic (PhSiO 1.5 ) 6 ligands (which give a charge of 12) that coordinate the Cu II 4containing core (which give a charge of + +8). Notably, for all early examples of such CLMS motifs, four additional bearers of positive charge (e.g., Na + + , Li + + , Cs + + ) were observed, which is logical to balance the charge of the CLMS. According to this logic, two Na cations in the composition of 1 are clearly not enough to balance the negative charge (from the silsesquioxane ligands), and the appearance of two [Cu I (dppe) 2 ] + + fragments becomes essential to form the final complex (Figure 2).

In turn, the silsesquioxane cage component of 2 is unprecedented. First, the nonacopper(II) silsesquioxane cage of 2 joins the very small family of nonanuclear CLMSs (the first and only known representative of that family was reported by some of us recently [START_REF] Bilyachenko | pin[END_REF] ). The anionic nonacopper cage of 2 possesses principal differences in its structure both from the compound described in Ref. [20] and from all other types of CLMSs. In the composition of 2, the two sandwich-like tetracopper fragments are joined by a central (ninth) Cu ion. Such a unique "fused" structure is possible because of the involvement of two types of silsesquioxane ligands: two cyclic (PhSiO 1.5 ) 6 and two acyclic (PhSiO 1.5 ) 4 ligands. The latter are the rarest type of silsesquioxane fragments and are able to coordinate metal ions in CLMSs.

To the best of our knowledge, the only observation of such a ligand in the composition of CLMS was reported by some of us for a Mn-containing silsesquioxane. [21] Similar to 1, the negative charge from the silsesquioxane matrix of 2 is balanced (along with Cu II ions of cage component) by four Na + + cations and two [Cu I (dppe) 2 ] + + fragments (Figure 3).

As we were interested in the further investigation of this nontrivial chemistry, we performed some additional experiments (Scheme 2). First, a change of the reactant ratio let us to isolate product 3, which is a rare instance of a compound that includes different (by their nuclearity) CLMS compositions in the same complex. This product 3 involves two types of com- ponents: (i) compound 2 and (ii) a hexacopper silsesquioxane with a cylinder-like structure (Figure 4).

Then, we tried to optimize the conditions of the synthesis of dppe-containing CLMSs. Previously, the syntheses of 1-3 were performed with heating to reflux because of the low solubility of dppe in ethanol at room temperature. The replacement of ethanol with an ethanol/toluene mixture (Scheme 2) allowed us to synthesize 2 at a lower temperature and to isolate product 4 (Figure 5) as an ethanol/toluene complex in a higher yield than 2 (46 vs. 17 %).

Then, we explored the suitability of other reaction systems to prepare structural analogues of 2. To our surprise, the replacement of ethanol by DMSO (Scheme 2) led to some reorganization of the structure. Isolated as a DMSO complex, 5 is similar (with regard to the silsesquioxane skeleton) to 2. However, the phosphine components present in 2 do not participate in the formation of 5. As a result, 5 may be described as a one-component analogue of 2, in which electroneutrality is reached by the involvement of two additional Na + + centers (Figure 6).

The elimination of a phosphine component if DMSO is used as the reaction medium was reproducible. Synthesis under conditions close to those used for the formation of 5 with the partial replacement of Na ions by Cs ions (Scheme 2) resulted in the isolation of the trimetallic Cu 9 Na 3 Cs 3 product 6, which also contained no dppe (Figure 7). To our delight, 6 differs from 5 by its ability to pack into a 2D coordination polymer net. We suggest that this could be because of the different location of the Cs ions in the structure of 6 (at "crown ether" positions, coordinated by six-membered siloxane rings) to the Na ions in 5. As a result, the coordination polymer 6, in comparison to 1-5, is much less soluble (1-5 are easily soluble in many polar organic solvents, except for short-chain alcohols).

Solid-state structures and topological analysis

Notably, as for globular and prismatic Cu CLMSs, [11b, 12-13] a bulky phenyl coating prevents the condensation of neighboring cages. Bonding between the alkaline atoms of the CLMS cage and the O atoms of silsesquioxanes of neighboring molecules is not observed in 1-6. At the same time, as some of us observed previously, the replacement of Na with Cs favors the formation of coordination polymers, [13] here through bridging solvent molecules. In 6, 2D polymers can be found with CLMSs connected through bridging DMSO molecules and fully occupied positions of Cs. The chains are also somewhat connected through partially occupied positions of Cs atoms to a quasi-sql net (in which CLMSs form a net with a square-planar topology).

In terms of the Cu nuclearity, all products reported here could be divided into three categories: tetra-(1), hexa-( 3), and nonanuclear complexes (2-6). A description of the topology of the first two types was given by some of us recently. [22] In turn, nonanuclear Cu clusters in terms of NDk-m notation [23] are 1,2,4 M9-1 clusters in which 1,2,4 are the coordination numbers of topologically nonequivalent nodes, M denotes a discrete cluster, 9 is the number of metal atoms in the cluster, and 1 is a classification number to distinguish topologically distinct clusters with equal NDk parameters (Figure 8). In these clusters Cu atoms realize three different types of connectivity with different orientations of the equatorial and axial planes for connected atoms. Notably, these types of clusters were not only absent in CLMSs synthesized previously but were not observed previously in other metal clusters (see the TTD collection at http://topospro.com/databases/ttd/).

Catalytic oxidation

Oxidation of alkanes and alcohols with peroxides

Polynuclear Cu derivatives constitute active reaction centers of certain oxidizing enzymes (e.g., oxidases, particulate methane monooxygenase (pMMO), a-hydroxylating monooxygenase). [24] Various Cu complexes are enzyme models, [25] and some Cu-catalyzed oxidations of cyclohexane have been reported. [25i] Recently, we found that copper silsesquioxanes catalyze the efficient oxidation of benzene, alkanes, and alcohols with peroxides. [START_REF] Bilyachenko | pin[END_REF]22,26] Here, we report on the interaction of 1 and 4 with peroxides in the presence of alkanes, RH, which afforded oxygenated products, namely, alkyl hydroperoxides, ROOH. Secondary alcohols have been oxidized catalytically into ketones.

Kinetic plots of the product accumulation in cyclohexane oxidation are presented in Figure 9. The dependence of the initial oxidation rate W 0 on the initial concentration of catalyst 4 and cyclohexane are shown in Figure 10. The heating of a solution of cyclohexanol and tert-butyl hydroperoxide (TBHP) in acetonitrile gave cyclohexanone in 72 % yield after 5 h (Figure 11). The oxidation of 1-phenylethanol afforded acetophenone in 76 % yield after 10 h (Figure 12).

We also tested 1 in the catalytic oxidation of cyclohexane and alcohols. The heating of a solution of cyclohexane (0.46 m) and H 2 O 2 (2 m) in acetonitrile in the presence of 1 (5 10 4 m) at 60 8C for 1 h gave cyclohexanol (0.039 m) and cyclohexanone (0.015 m) with a total turnover number (TON) of 108. Notably, in this case the addition of HNO 3 (0.05 m) led to a sharp decrease of the product yield (0.004 and 0.00027 m of cyclohexanol and cyclohexanone, respectively). The reaction of cyclohexanol (0.38 m) with TBHP (1.2 m) catalyzed by 1 (5 10 4 m) at 60 8C after 3 h gave cyclohexanone (70 %). Acetophenone in 94 % yield was obtained under the same conditions from 1phenylethanol (0.49 m).

Catalytic oxidation of alcohols and amines to produce amides

Previously, we reported the ability of CLMSs to catalyze the oxidative formation of amides from alcohols. [6c, 22, 28] This reaction could be performed using Cu, [29] Fe, [30] or Zn, [31] but the catalyst loading could not be decreased below 1 mol %. With the use of CLMSs, this reaction was efficient with dramatically lower loadings (down to 100 ppm of Cu [22] and 500 ppm of Fe [6c, 28] ). As 1 and 4 present really atypical structures, their catalytic activity was also evaluated in the transformation of benzylic alcohol into benzamide. Reactions were performed using as low as 100 ppm of Cu and calcium carbonate 99.995 % pure to ensure that Cu was the only metal responsible for the catalysis. Thus, benzylic alcohol was used in the presence of various primary and secondary amines with TBHP as an oxidant in acetonitrile heated to reflux for 24 h (Table 1). Notably, the concomitant use of the ammonium salt and calcium carbonate was compul-sory to release free amine slowly in the reaction mixture to observe good conversions. Gratifyingly, even though a really low amount of Cu was used, the desired amides could be isolated in good yields (64-84 %). Primary amines (Table 1, entries 1-6) were converted slightly more easily than secondary ones (Table 1, entries 7-12). CLMSs 1 and 4 behaved similarly under the reaction conditions and excellent TONs and turnover frequencies (TOFs) up to 8400 and 350 h 1 , respectively, could be obtained. In comparison, if 2 mol % of CuO was used in the original study on Cu-catalyzed oxidative amidation, [29a] the TON and TOF values did not exceed 44 and 11 h 1 , respectively. Hence, low-loading CLMS catalysis was extremely efficient.

Conclusion

We present a detailed study of a simple new method to prepare cage-like metallasilsesquioxanes. This method implies the use of a bidentate phosphine (1,2-bis(diphenylphosphino)ethane; dppe) ligand as a key reagent in the formation of Cuand Na-based cage products with different nuclearities (Cu 4 Na 2 and Cu 9 Na 4 ). The involvement of two types of ligands (silsesquioxane and phosphine) is responsible for an unprecedented redistribution of Cu ions between two types of ionic components (cage Cu,Na-silsesquioxane and Cu(dppe) 2 ) to form the complex product. This reaction was sensitive to the solvent system, which allowed us to isolate additional examples of fascinating cage silsesquioxane architectures (Cu 9 Na 6 and Cu 9 Na 3 Cs 3 ). The first example of the evaluation of complexes of this family as precatalysts in homogeneous catalysis revealed the high activity of these complexes in oxidations with H 2 O 2 and tert-butyl hydroperoxide. In turn, the catalytic formation of benzamides was promising as a Cu loading as low as 100 ppm with the use of 1 and 4 allowed us to reach a turnover number up to 8400 and a turnover frequency up to 350 h 1 .

A further focus on the synthesis of these cage-like metallasilsesquioxanes will no doubt lead to the discovery of currently unknown structural types of these cage compounds as well as the new possibilities for their catalytic applications.

Experimental Section

Chemicals PhSi(OEt) 3 (98 %) and dppe (99 %) were purchased from Sigma-Aldrich. Solvents were purchased from Acros.

Synthesis

Compound 1: PhSi(OEt) 3 (1 g, 4.16 mmol) and NaOH (0.194 g, 4.85 mmol) were heated to reflux in ethanol (60 mL) for 1.5 h. CuCl 2 (0.28 g, 2.08 mmol) was added, and the resulting blue mixture was stirred without heating for 1 h. Afterwards, dppe (0.414 g, 1.04 mmol) was added at once, and the resulting mixture was heated under reflux for 3 h. The hot solution was filtered to remove the precipitate. The crystallization of the filtrate over 1 week gave a crystalline material, and several single crystals of which were used for XRD analysis (1•4 EtOH, see the Supporting Information for details). Yield 0.40 g (42 %). Elemental analysis calcd (%) for [(PhSiO 1.5 ) 12 (CuO) 4 (NaO 0.5 ) 2 ][Cu(dppe) 4 ] 2 (3651.23): Si 9.23, Cu 10.44, Na 1.26, P 6.79; found (for vacuum-dried sample): Si 9.17, Cu 10.38, Na 1.20, P 6.72.

Compound 2: PhSi(OEt) 3 (1 g, 4.16 mmol) and NaOH (0.216 g, 5.40 mmol) were heated to reflux in ethanol (60 mL) for 1.5 h. CuCl 2 (0.28 g, 2.08 mmol) was added, and the resulting blue mixture was stirred without heating for 1 h. Afterwards, dppe (0.414 g, 1.04 mmol) was added at once, and the resulting mixture was heated to reflux for 3 h. The hot solution was filtered to remove the precipitate. The crystallization of filtrate over 1 week gave a crystalline material, and several single crystals were used for XRD analysis ( 2 Compound 3: PhSi(OEt) 3 (1 g, 4.16 mmol) and NaOH (0.188 g, 4.70 mmol) were heated to reflux in ethanol (60 mL) for 1.5 h. CuCl 2 (0.28 g, 2.08 mmol) was added, and the resulting blue mixture was stirred without heating for 1 h. Afterwards, dppe (0.414 g, 1.04 mmol) was added at once, and the resulting mixture was heated to reflux for 3 h. The hot solution was filtered to remove the precipitate. The crystallization of the filtrate over 1 week gave a crystalline material, several single crystals of which were used for XRD analysis (3•12 EtOH•2 H 2 O), see the Supporting Information for details). Yield 0.12 g (13 %). Elemental analysis calcd (%) for [(PhSiO 1.5 ) 12 (PhSiO 1.5 ) 8 (CuO) 8 (NaO 0.5 ) 4 {Cu(O 0.5 ) 4 }][Cu(dppe) 2 ] 2 [(PhSi-O 1.5 ) 12 (CuO) 6 ] (7187.93): Si 12.50, Cu 15.03, Na 1.28, P 3.45; found (for vacuum-dried sample): Si 12.41, Cu 14.92, Na 1.22, P 3.39.

Compound 4: PhSi(OEt) 3 (1 g, 4.16 mmol) and NaOH (0.216 g, 5.40 mmol) were heated to reflux in ethanol (60 mL) for 1.5 h. CuCl 2 (0.28 g, 2.08 mmol) was added, and the resulting blue mixture was stirred without heating for 1 h. Afterwards, dppe (0.414 g, 1.04 mmol) dissolved in toluene (45 mL) was added at once, and the resulting mixture was stirred for 2 h. The solution was filtered to remove the precipitate. The crystallization of the filtrate over 2 weeks gave a crystalline material, and several single crystals were Compound 6: PhSi(OEt) 3 (1 g, 4.16 mmol) and NaOH (0.216 g, 5.40 mmol) were heated to reflux in ethanol (50 mL) for 1.5 h. CuCl 2 (0.28 g, 2.08 mmol) was added, and the resulting blue mixture was stirred without heating for 1 h. Afterwards, dppe (0.414 g, 1.04 mmol) and DMSO (60 mL) were added at once. The resulting mixture was heated to reflux along with the simultaneous distillation of the solution to remove ethanol completely from the reaction mixture. When 45 mL of distillate was collected, CsF (0.43 g, 2.83 mmol) was added at once to the mixture. The resulting solution was heated to reflux for an additional 3 h then cooled to RT and filtered from the precipitate. The crystallization of the filtrate over 2 weeks gave a crystalline material, and several single crystals were used for XRD analysis (6•9 DMSO, see the Supporting Information for details). Yield 0.12 g (15 %). Elemental analysis calcd (%) for [(PhSiO 1.5 ) IR spectroscopy of the bulky samples of crystalline materials of 1-6 were performed by using a Shimadzu IRPrestige-21 FTIR spectrometer (KBr) in the 600-4000 cm 1 range. IR spectra are given in the Supporting Information.

Oxidation of alcohols and alkanes

The reactions of alcohols were performed in air by using thermostated cylindrical Pyrex vessels with vigorous stirring and MeCN as the solvent. The substrate was then added, and the reaction started on introduction of the oxidant in one portion. The concentrations of products obtained in the oxidation of 1-phylethanol after certain time intervals were measured by using 1 H NMR spectroscopy (solutions in [D 6 ]acetone; Bruker AMX-400 instrument, 400 MHz). In the oxidation of cyclohexanol, concentrations of the substrate and products were measured by chromatography as described below for the oxidation of cyclohexane.

Oxidations of alkanes were performed analogously. Catalysts 1 or 4 and the cocatalyst (HNO 3 ) were introduced into the reaction mixture in the form of stock solutions in acetonitrile. The alkane was then added, and the reaction started on introduction of H 2 O 2 in one portion. (CAUTION: The combination of air or O 2 and H 2 O 2 with organic compounds at elevated temperatures may be explo-sive!) The reaction solutions were analyzed by using GC after the addition of nitromethane as a standard compound. A LKhM-80-6 instrument was used (columns 2 m with 5 % Carbowax 1500 on 0.25-0.315 mm Inerton AW-HMDS; carrier gas Ar) to measure concentrations of cyclohexanol and cyclohexanone. Samples of reaction solutions were in some cases analyzed twice: before and after their treatment with PPh 3 . This method (an excess of solid PPh 3 is added to the samples 10-15 min before the GC analysis) proposed previously by one of us [27] allows us to detect alkyl hydroperoxides and to measure also the real concentrations of all three products (alkyl hydroperoxide, alcohol, and aldehyde or ketone) present in the reaction solution because alkyl hydroperoxides are usually decomposed in the gas chromatograph to produce mainly the corresponding alcohol and ketone. It is necessary to emphasize that the PPh 3 is added mainly to compare intensities of cyclohexanol and cyclohexanone peaks and not to reduce the remaining H 2 O 2 . The comparison gives us the real concentration of cyclohexyl hydroperoxide in the reaction mixture. In our kinetic studies described below, to determine the initial reaction rate W 0 we measured the concentrations of cyclohexanone and cyclohexanol only after the reduction of the reaction mixture with PPh 3 , which gives the precise concentration of a sum of the oxygenates. Blank experiments with cyclohexane showed that in the absence of 1 and 4 no products were formed.

Amides synthesis

General considerations: All reagents were purchased from Aldrich Chemical Co., Fluka, and Alfa Aesar and used without further purification. Analyses were performed at the 'Plateforme Technologique Laboratoire de Mesures Physiques' (IBMM, Universit de Montpellier). 1 H NMR spectra were recorded by using a Bruker Avance DPX 300 MHz spectrometer. Chemical shifts are reported in ppm and referenced to the solvent peak (CDCl 3 at 7.26 ppm). Data are reported as s = singlet, d = doublet, t = triplet, q = quadruplet, qt = quintuplet, sept = septuplet, and m = multiplet; coupling constants in Hz; integration. 13 C NMR spectra were recorded by using a Bruker Avance AM 126 MHz spectrometer. Chemical shifts are reported in ppm and referenced to the solvent peak (CDCl 3 at d = 77.16 ppm).

General procedure: Into a sealed tube were added successively amine hydrochloride (0,5 mmol), CaCO 3 (50.1 mg, 0.5 mmol), CH 3 CN (1 mL), 1 (20 mL of a solution of 3.0 mg of 1 in 2 mL of CH 3 CN) or 3 (20 mL of a solution of 1.8 mg of 3 in 2 mL of CH 3 CN), benzylic alcohol (105 mL, 1.0 mmol), and TBHP (5.5 m in nonane, 225 mL, 1.25 mmol). The mixture was stirred at 80 8C for 2 h, and TBHP (5.5 m in nonane, 225 mL, 1.25 mmol) was added again to the mixture. After 22 h at 80 8C, the mixture was cooled to RT, and 1 n HCl and AcOEt were added. The mixture was extracted twice with AcOEt, and the combined organic phases were washed with a saturated solution of NaHCO 3 and brine and concentrated under reduced pressure. To remove the excess of benzylic alcohol, H 2 O (80 mL) was added and evaporated under reduced pressure. The crude product was then purified using silica gel chromatography using gradients of cyclohexane/AcOEt to yield the pure compounds.

Figure 1 .

 1 Figure 1. Structures of (PhSiO 1.5 ) 12 (CuO) 4 (NaO 0.5 ) 2 (Cu) 2 (dppe) 4 (1; solvating molecules of EtOH are omitted for clarity) and (PhSiO 1.5 ) 12 (PhSiO 1.5 ) 8 (CuO) 8 -(NaO 0.5 ) 4 {Cu(O 0.5 ) 4 }(Cu) 2 (dppe) 4 (2; solvating molecules of EtOH are omitted for clarity).

Figure 2 .

 2 Figure 2. Copper silsequioxane cage in 1. Color code: Si = light yellow, O = red, Cu = blue, Na = yellow.

Figure 3 .

 3 Figure 3. Copper silsequioxane cage in 2. Color code: Si = light yellow, O = red, Cu = blue, Na = yellow.

Figure 4 .

 4 Figure 4. Structures of [(PhSiO 1.5 ) 12 (PhSiO 1.5 ) 8 (CuO) 8 (NaO 0.5 ) 4 {Cu(O 0.5 ) 4 }][Cu(dppe) 2 ] 2 [(PhSiO 1.5 ) 12 (CuO) 6 ] (3; solvating molecules of water and EtOH are omitted for clarity).

Figure 5 .

 5 Figure 5. Structures of [(PhSiO 1.5 ) 12 (PhSiO 1.5 ) 8 (CuO) 8 (NaO 0.5 ) 4 {Cu(O 0.5 ) 4 }] [Cu(dppe) 2 ] 2 (4; solvating molecules of water and EtOH are omitted for clarity).

Figure 6 .

 6 Figure 6. Structures of [(PhSiO 1.5 ) 12 (PhSiO 1.5 ) 8 (CuO) 8 (NaO 0.5 ) 4 {Cu(O 0.5 ) 4 }Na 2 ] (5; solvating molecules of DMSO are omitted for clarity).

Figure 7 .

 7 Figure 7. Top left: molecular structure of [(PhSiO 1.5 ) 12 (PhSiO 1.5 ) 8 (CuO) 8 (NaO 0.5 ) 3 (CsO 0.5 ){Cu(O 0.5 ) 4 }Cs 2 ] (6; solvating molecules of DMSO are omitted for clarity). Top right: ChemDraw structure of 6. Bottom: 2D coordination polymer structure of 6.

Figure 8 .

 8 Figure 8. a) Nonacopper-containing clusters connected by bridging atoms in CLMSs 2-6 and b) scheme of cluster connectivity.

Figure 9 .

 9 Figure 9. Top: general scheme for the oxidation of cyclohexane. Bottom: accumulation of cyclohexanol (curve 1) and cyclohexanone (curve 2) in the course of cyclohexane oxidation with H 2 O 2 in acetonitrile in air. Conditions: 4, 5 10 4 m; cyclohexane, 0.46 m; H 2 O 2 (50 % aqueous, 2 m). A) 20 8C; in the presence of HNO 3 , 0.05 m; maximum yield of oxygenates is 20 % after 1 h; TON = 182. B) 60 8C; in the presence of HNO 3 , 0.1 m. C) 60 8C; in the absence of HNO 3 .In all cases concentrations of the oxygenates were measured after the addition of PPh 3 to the reaction sample.[27]

Figure 10 .

 10 Figure 10. Oxidation of cyclohexane with H 2 O 2 in acetonitrile in air. Conditions: Catalyst 4, 5 10 4 m; H 2 O 2 (50 % aqueous, 2m) at 60 8C in the presence of HNO 3 , 0.05 m. Concentrations of the oxygenates were measured after the addition of PPh 3 to the reaction sample.

Figure 11 .

 11 Figure 11. Accumulation of cyclohexanone (curve 1) in the course of cyclohexanol (0.38 m) oxidation with TBHP (70 % aqueous, 1.2 m) catalyzed by 4 (5 10 4 m) in acetonitrile in air at 60 8C. Maximum yield of oxygenates is 72 % after 5 h; TON = 550. Curve 2: the same reaction in the presence of HNO 3 , 0.05 m. Concentrations of the ketone were measured after the addition of PPh 3 to the reaction sample.

Figure 12 .

 12 Figure 12. Top: general scheme for the oxidation of 1-phenylethanol. Bottom: accumulation of acetophenone (curve 2) and 1-phenylethanol consumption (curve 1) in the course of 1-phenylethanol (0.5 m) oxidation with TBHP (70 % aqueous, 1.2 m) catalyzed by 4 (5 10 4 m) in acetonitrile in air at 60 8C in the presence of HNO 3 , 0.05 m.

  •8 EtOH•2 H 2 O, see the Supporting Information for details). Yield 0.18 g (17 %). Elemental analysis calcd (%) for [(PhSiO 1.5 ) 12 (PhSiO 1.5 ) 8 (CuO) 8 (NaO 0.5 ) 4 {Cu(O 0.5 ) 4 }][Cu(dppe) 2 ] 2 (5160.39): Si 10.89, Cu 13.55, Na 1.78, P 4.80; found (for vacuumdried sample): Si 10.81, Cu 13.46, Na 1.60, P 4.71.

  [a] Reaction conditions: amine•HCl (0.5 mmol), benzyl alcohol (1.0 mmol), CaCO 3 (99.995 % pure; 0.5 mmol), TBHP (5.5 m in nonane, 2.5 mmol), 1 or 4 (100 ppm of Cu), CH 3 CN (1 mL), 80 8C, 24 h. Isolated yields are given. [b] TON = (mmol of product)/(mmol of Cu). [c] TOF = TON/(reaction time).used for XRD analysis (4•6 EtOH•2 H 2 O•2 C 7 H 8 , see the Supporting Information for details). Yield 0.49 g (46 %). Elemental analysis calcd (%) for [(PhSiO 1.5 ) 12 (PhSiO 1.5 ) 8 (CuO) 8 (NaO 0.5 ) 4 {Cu(O 0.5 ) 4 }][Cu(dppe) 2 ] 2 (5160.39): Si 10.89, Cu 13.55, Na 1.78, P 4.80; found (for vacuumdried sample): Si 10.81, Cu 13.46, Na 1.60, P 4.71.Compound 5: PhSi(OEt) 3 (1 g, 4.16 mmol) and NaOH (0.216 g, 5.40 mmol) were heated to reflux in ethanol (50 mL) for 1.5 h. CuCl 2 (0.28 g, 2.08 mmol) was added, and the resulting blue mixture was stirred without heating for 1 h. Afterwards, dppe (0.414 g, 1.04 mmol) and DMSO (60 mL) were added at once. The resulting mixture was heated to reflux along with the simultaneous distillation of the solution to remove ethanol completely from the reaction mixture. When approximately 45 mL of distillate was collected, the mixture was cooled to RT and filtered to remove the precipitate. The crystallization of the filtrate over 2 weeks gave a crystalline material, and several single crystals were used for XRD analysis (5•8 DMSO, see the Supporting Information for details). Yield 0.14 g (19 %). Elemental analysis calcd (%) for [(PhSiO 1.5 ) 12 (PhSi-O 1.5 ) 8 (CuO) 8 (NaO 0.5 ) 4 {Cu(O 0.5 ) 4 }Na 2 ] (3485.62): Si 16.12, Cu 16.41, Na 3.96, P 0.0; found (for vacuum-dried sample): Si 16.06, Cu 16.32, Na 3.870, P 0.0.

Table 1 .

 1 Oxidative amidation at low catalyst loading.[a]

	Entry	Amide	Catalyst	Yield [%]	TON/TOF [h 1 ] [b,c]
	1		1	76	7600/317
	2		4	77	7700/321
	3		1	74	7400/308
	4		4	68	6800/283
	5		1	82	8200/342
	6		4	83	8300/346
	7		1	67	6700/279
	8		4	66	6600/275
	9		1	64	6400/267
	10		4	68	6800/283
	11		1	77	7700/321
	12		4	84	8400/350

ChemCatChem 2017, 9, 4437 -4447 www.chemcatchem.org 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemcatchem.org 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Acknowledgements

This work has been supported by the Russian Foundation for Basic Research (Grants 16-03-00254; 16-53-150008; 16-29-

Conflict of interest

The authors declare no conflict of interest.