Almasi Miroslav
email: miroslav.almasi@upjs.sk

Jozef Pavol

Safarik

Pavla Jozefa

Miroslav Almasi

Nikolas Király

Prof Vladimír Zeleňák

Dr Sandrine Mária Vilková

Dr. Bourrelly

Dr Miroslav Almáši

Mária Vilková

Sandrine Bourrelly

Cadmium(II) Amorphous Metal-Organic Frameworks (aMOFs): Study of Activation Process and High-pressure Adsorption of Greenhouse Gases

Keywords: amorphous metal-organic frameworks, gas adsorption, greenhouse gasses, carbon dioxide, methane amorphous metal-organic frameworks, freeze-drying process, gas adsorption, greenhouse gasses, carbon dioxide, methane

Two novel amorphous metal-organic frameworks (aMOFs) with chemical composition -14) built up Zn(II) and Cd(II) ions and extended tetrahedral tetraazotetracarboxylic acid (H 4 MTA) as linker were prepared and characterised. Nitrogen adsorption measurements were performed on as-synthesized (AS), ethanol exchanged (EX) and freeze-dried (FD) materials at different activation temperatures 60, 80, 100, 120, 150 and 200°C to obtain the best textural properties. The largest surface areas of 830 m 2 .g -1 for UPJS-13 (FD) and 1057 m 2 .g -1 for UPJS-14 (FD) were calculated from nitrogen adsorption isotherms for freeze-dried materials activated at mild activation temperature (80°C). Subsequently, the prepared compounds were tested as adsorbents of greenhouse gasses, carbon dioxide and methane, measured at high pressures. The maximal adsorption capacities were 30.01 wt. % of CO 2 and 4.84 wt. % of CH 4 for UPJS-13 (FD) and 24.56 wt. % of CO 2 and 6.38 wt. % of CH 4 for UPJS-14 (FD) at 20 bar and 30°C.

Introduction

Metal-organic frameworks (MOFs) are one of the most exciting classes of porous materials to be discovered in the past decades. MOFs are crystalline materials constructed from organic molecules (linkers) coordinated to metal ions or clusters to form interesting polymeric frameworks with a larger surface area and the advantage of pore size changing. Due to the porosity and tunable textural properties, MOF materials find applications in gas storage and separation [START_REF] Qian | MOF-based membranes for gas separations[END_REF][START_REF] Ghazvini | Investigation of the MOF adsorbents and the gas adsorptive separation mechanisms[END_REF], heterogeneous catalysis [START_REF] Goetjen | Metal-organic framework (MOF) materials as polymerisation catalysts: A review and recent advances[END_REF][START_REF] Konnerth | Metal-organic framework (MOF)-derived catalysts for fine chemical production[END_REF], drug delivery [START_REF] Cao | Metal-organic framework (MOF)-based drug delivery[END_REF][START_REF] Wang | Metalorganic frameworks for stimuli-responsive drug delivery[END_REF], magnetic refrigeration [START_REF] Kim | Research trend of metal-organic frameworks for magnetic refrigeration materials application[END_REF][START_REF] Zeleňák | Large and tunable magnetocaloric effect in gadolinium-organic framework: Tuning by solvent exchange[END_REF], sensing [START_REF] Wang | Metal-organic frameworks for QCM-based gas sensors: A review[END_REF][START_REF] Li | Functional metal-organic frameworks as effective sensors of gases and volatile compounds[END_REF] and energy storage [START_REF] Capková | Metal-organic framework MIL-101(Fe)-NH2 as an efficient host for sulphur in long-cycle Li-S batteries[END_REF][START_REF] Cai | MOF-derived conductive carbon nitrides for separator-modified Li-S batteries and flexible supercapacitors[END_REF]. A small but growing number of noncrystalline MOFs are steadily capturing scientific interest, though it is only recently that well-characterised examples of amorphous metal-organic frameworks (aMOFs) have started to appear [START_REF] Bennett | Liquid, glass and amorphous solid states of coordination polymers and metal-organic frameworks[END_REF][START_REF] Ohtsu | Amorphousamorphous transition in a porous coordination polymer[END_REF][START_REF] Halder | Structure and properties of dynamic metal-organic frameworks: A brief accounts of crystalline-to-crystalline and crystalline-to-amorphous transformations[END_REF]. aMOFs retain the basic building blocks and connectivity of their crystalline counterparts, though they lack any long-range periodic order. Amorphous MOFs offer many exciting opportunities for practical application, either as novel functional materials themselves or facilitating other processes, though the domain is mostly unexplored [START_REF] Bennett | Amorphous metal-organic frameworks[END_REF]. Published articles dealing with aMOFs have been demonstrated to be of potential in several fields, e.g. catalysis [START_REF] Zhang | Facile synthesis of highly efficient amorphous Mn-MIL-100 catalysts: Formation mechanism and structure changes during application in CO oxidation[END_REF][START_REF] Ma | Heterogeneous amorphous Cu-MOF-74 catalyst for C-N coupling reaction[END_REF][START_REF] Li | Synthetic chemistry and multifunctionality of an amorphous Ni-MOF-74 shell on a Ni/SiO2 hollow catalyst for efficient tandem reactions[END_REF][START_REF] Zhou | Amorphous intermediate derivative from ZIF-67 and its outstanding electrocatalytic activity[END_REF], drug delivery [START_REF] Orellana-Tavra | Amorphous metal-organic frameworks for drug delivery[END_REF][START_REF] Wu | Packaging and delivering enzymes by amorphous metal-organic frameworks[END_REF][START_REF] Suresh | Enhanced drug delivery by dissolution of amorphous drug encapsulated in a water unstable metal-organic framework (MOF)[END_REF][START_REF] Liu | Recent development of amorphous metal coordination polymers for cancer therapy[END_REF][START_REF] Orellana-Tavra | Biocompatible, crystalline, and amorphous bismuth-based metal-organic frameworks for drug delivery[END_REF], supercapacitors [START_REF] Yang | Facile synthesis of amorphous UiO-66 (Zr-MOF) for supercapacitor application[END_REF][START_REF] Niu | Amorphous MOF introduced N-doped graphene: An efficient and versatile electrocatalyst for zinc-air battery and water splitting[END_REF][START_REF] Xiu | Electrical bistability in a metal-organic framework modulated by reversible crystalline-to-amorphous transformations[END_REF] and irreversible harmful substance capture [START_REF] Bennett | Ball-milling-induced amorphisation of zeolitic imidazolate frameworks (ZIFs) for the irreversible trapping of iodine[END_REF][START_REF] Zhang | Amorphous Fe/Mn bimetal-organic frameworks outer and inner structural designs for efficient arsenic(III) removal[END_REF].

To study the porosity and textural properties of porous materials is necessary to activate them and thus obtain an open structure. A typical activation process is a thermal activation, in which the process of releasing solvent molecules is caused by heat. This activation way often leads to the shrink or collapse of the polymeric frameworks causing a decrease of material´s porosity or non-porosity. To reduce the activation temperature, a solvent exchange process is also used as a tool, in which high-boiling solvents are exchanged for low-boiling solvents and the thermal activation of porous material occurs at a lower temperature. The freeze-drying process, also known as lyophilisation or cryodesiccation, is a dehydration/desolvation process performed at low temperature, which involves freezing the material with guest molecules and subsequent sublimating the guests under low temperature and low pressure. Lyophilisation is a commercial process preliminary used in biological, biomedical processing, fine food production and preservation. The freeze-drying process has also found an application in the activation of porous materials [START_REF] Zhang | Fundamentals of controlled freezing for ice-templated porous materials, Ice templating and freeze-drying for porous materials and their applications[END_REF]. Large specific surface area and better textural properties can be obtained by exchanging of high boiling point solvents (N,N´-dimethylformamide (153°C), N,N´-diethylformamide (176°C), N,N´-dimethylacetamide (165°C), dimethylsulfoxide (189°C) etc.), that are typically located in the pores of as-synthesized material by the solvents with high melting point and high vapour pressure (benzene (5.5°C, 75 mmHg at 20°C), cyclohexane (4°C, 77 mmHg at 20°C), tert-butyl alcohol (23°C, 31 mmHg at 20°C), o-xylene (-23°C, 7 mmHg at 20°C) and compressed CO2) followed by evacuation at low temperature. For the organic nature of porous coordination polymers, many MOFs exhibit surface areas that are only a small fraction of predicted/calculated surfaces based on their crystal structures. It was shown that a freeze-drying process could achieve a higher surface area and the desired features of porous materials. For example, freeze-dried compounds [Cu2(MTB)(H2O)2]•6DEF•2H2O and [Cu2(MTC)(H2O)2]•14DMF•5H2O, which also contain tetrahedral linkers (MTB = methanetetrabenzoate, MTC = methanetetra(biphenyl-pcarboxylate)) exhibited surface areas of 526 m 2 .g -1 and 791 m 2 .g -1 for evacuated materials (activation temperature 60°C) and 1560 m 2 .g -1 , 1020 m 2 .g -1 for freeze-dried samples, respectively [START_REF] Ma | Freeze drying significantly increases permanent porosity and hydrogen uptake in 4,4-connected metal-organic frameworks[END_REF]. As is obvious from described values, lyophilisation is an efficient process for activating porous materials that can increase the surface area three times compared to standard thermal activation.

Global temperature has been rising since the first industrial revolution in the early 19 th century, due to the emissions of large quantities of greenhouse gases. The main greenhouse gases are carbon dioxide and methane, but also nitrogen oxides and water vapour. Carbon

SBET = 2880 m 2 .g -1 )
, which can store up 90 wt.% CO2 [START_REF] Kim | Cooperative carbon capture and steam regeneration with tetraamine-appended metal-organic frameworks[END_REF] and amine grafted silica materials [START_REF] Zeleňák | Phase evolution during one-pot synthesis of amine modified mesoporous silica materials: Preparation, properties, carbon dioxide adsorption[END_REF][START_REF] Anyanwu | Amine-grafted silica gels for CO2 capture including direct air capture[END_REF][START_REF] Zhao | A novel blended amine functionalised porous silica adsorbent for carbon dioxide capture[END_REF]. In order to avoid CO2-intensive energy processes, CO2 molecules must be captured in porous materials by physisorption as a chemisorption process. However, physisorption brings new challenges, because there is a lack of selectivity for CO2 because no chemical bond is formed or when the functional groups are tuned to increase selectivity, the absorption of CO2 is limited.

In the present study, extended tetrahedral nitrogen-rich tetraazo-tetracarboxylic acid (H4MTA) was prepared by seven-step organic synthesis and used as a linker in the preparation of two novel aMOFs containing Zn(II) (UPJS-13) and Cd(II) (UPJS-14) ions. After characterisation, the textural properties of prepared materials were studied by nitrogen adsorption at -196°C on as-synthesized (AS), ethanol exchanged (EX) and freeze-dried (FD)

samples. The specific surface area (SBET) depended on the sample pretreatment (AS < EX < FD)

and the activation temperature (60-200°C). The highest SBET values were obtained for FD materials after activation process at 80°C: UPJS-13 (EX) 830 m 2 .g -1 and UPJS-14 (EX) 1057 m 2 .g -1 . After finding the best activation conditions, the materials were tested as adsorbents of greenhouse gases in high-pressure adsorption measurements of carbon dioxide and methane up to 20 bar.

Experimental

Synthetic part

Used chemicals

All chemicals used in the synthesis of methanetetrayltetrakis(benzene-4,1diyl)tetrakis(aza))tetrakis(methan-

Synthesis of H4MTA

H4MTA linker was prepared by seven-step organic synthesis according to the reactions presented in Fig. 1 and procedures described below. Reaction conditions were chosen and modified from the literature procedures [START_REF] Almáši | Synthesis, characterisation and sorption properties of zinc(II) metal-organic framework containing methanetetrabenzoate ligand[END_REF][START_REF] Klumpen | Microporous organic polyimides for CO2 and H2O capture and separation from CH4 and N2 mixtures: interplay between porosity and chemical function[END_REF][START_REF] Shukla | Cardanol benzoxazinesinterplay of oxazine functionality (mono to tetra) and properties[END_REF][START_REF] Gonda | Synthesis and biological activity of sphingosines with integrated azobenzene switches[END_REF].

Synthesis of 4-tritylaniline (1)

4-tritylaniline was prepared by the condensation reaction of triphenylmethanol and aniline. Triphenylmethanol (20 g; 77 mmol) was dissolved in glacial acetic acid (230 cm 3 ) and to this solution aniline (11 cm 3 ; 121 mmol) was added. Finally, the reaction mixture was acidified with concentrated HCl (38.5 cm 3 ) and refluxed at 130°C for 48 h. Subsequently, the reaction mixture was cooled to ambient temperature and diluted in water (1.5 dm 3 ). The resulting precipitate of 1 was filtered off under reduced pressure using a Buchner funnel. The filter cake was washed several times with water to remove residual acetic and hydrochloric acid. The washed product was placed in an oven and dried at 70°C overnight to yield 18.

Synthesis of tetrakis(phenyl)methane (2)

The tetrakis(phenyl)methane core was prepared by deamination reaction of 4tritylaniline. 1 (10 g; 30 mmol) was dispersed in N,N´-dimethylformamide (DMF, 125 cm 3 ) and the solution was acidified by dropwise addition of concentrated sulphuric acid (62.5 cm 3 ).

Sulphuric acid protonated the primary amine of 1, causing its complete dissolution in DMF.

Subsequently, the reaction mixture was cooled with the solution of ice/sodium chloride to ≈ -10°C, and then isopentyl nitrite (15 cm 3 ; 112.5 mmol) was added dropwise. After the addition of isopentyl nitrite, the solution was stirred for 1 h at ≈ -10°C. Hypophosphorous acid (30 cm 3 ; 0.5 mol) was added dropwise after the mentioned reaction time, which catalysed the azo bond cleavage of the intermediate. During the addition of H3PO2, the release of nitrogen was observed and a precipitate of 2 was formed in the mixture.

Preparation of tetrakis(p-aminophenyl)methane (4)

The aim of this synthetic step was the reduction reaction of nitro to an amino group. The reduction was carried out by two synthetic procedures using different catalysts and reducing agents: hydrazine/RaNi or Pd/C and hydrogen, respectively.

Synthesis using hydrazine/RaNi: The reaction mixture of 3 (0.3 g; 0.9 mmol), hydrazine monohydrate (N2H4•H2O; 0.4 g; 8 mmol; 50% aqueous solution) and Raney nickel (Ra/Ni; 10 g) in THF (50 cm 3 ) was refluxed for 12 hours. The reaction mixture was hot filtered under reduced pressure to remove the heterogeneous catalyst and washed with THF. The filtered solution was evaporated under vacuum until the first visible crystals of the product were formed. The crystallised product 4 was filtered off, slightly washed with ethanol and finally purified from hot ethyl acetate (yield: 0.18 g, 78% based on 3).

Synthesis using Pd/C and hydrogen: 3 (0.5 g; 0.99 mmol) was dissolved in methanol (20 cm 3 ) and heterogeneous Pd/C catalyst (0.1 g; 0.05 mmol) was added to the solution in small portions. The reaction mixture was stirred vigorously and the hydrogen atmosphere was created as follows: the pressure in the system was reduced by a vacuum pump until boiling of the methanol occurred and hydrogen was introduced into the system. This procedure was repeated four times. Subsequently, the reaction mixture was stirred for 24 h in a reducing atmosphere of hydrogen at room temperature. The product was filtered off, washed with methanol and dried under vacuum. 4 was purified by column chromatography on silica gel using a solvent mixture of DCM: MeOH (7: 1, v/v) as mobile phase (yield: 0.27 g, 71% based on 3).

Preparation of ethyl 4-nitrosobenzoate (5)

2.5 g (15 mmol) of ethyl 4-aminobenzoate was dissolved in 50 cm 3 of dichloromethane (DCM) and solution of oxone (potassium peroxymonosulfate, KHSO5•0.5KHSO4•0.5K2SO4) 20.3 g (66 mmol) in 200 cm 3 of deionised water was added. The reaction mixture was stirred at room temperature for 2 h. After the colour change from colourless to green, the mixture was extracted with CH2Cl2 and washed with H2O several times. The organic layer was further washed with 30 cm 3 of 1M HCl and 40 cm 3 of NaHCO3 saturated aqueous solution. The suspension was extracted with CH2Cl2 (4 x 50 cm 3 ) and dried with Na2SO4. The organic layers were collected and the solvent was removed under reduced pressure to form the light-yellow product of 5 (yield 2.03 g, 75% based on ethyl 4-aminobenzoate). 1 H NMR (600 MHz, CDCl3)  (ppm): 1.24 (3H, t, J = 7.1 Hz), 4.14 (2H, q, J = 7.1 Hz), 8.07 (2H, ddd, J = 8.5, 1.6, 0.5 Hz), 8.13 (2H, ddd, J = 8.5, 1.4, 0.5 Hz). Elemental analysis for C9H9NO3 (179.17 g.mol -

Preparation of methanetetrayltetrakis(benzene-4,1-diyl)tetrakis(aza))tetrakis (methan-1-yl-1-yliden)tetrabenzoic acid ethyl ester (Et4MTA) (6)

Et4MTA was prepared by the condensation reaction of ethyl 4-nitrosobenzoate and tetrakis(p-aminophenyl)methane.

Solution of 5 (1.6 g, 9 mmol) in 40 cm 3 of acetic acid was added dropwise during 10 min time period in solution of 4 (0.7g, 1.8 mmol) dissolved in 40 cm 3 of glacial acetic acid and 50 cm 3 of extra dry methanol under nitrogen. The reaction mixture was stirred at laboratory temperature in an inert atmosphere of nitrogen for 2 days. Formed orange suspension of 6 was filtered off, washed with saturated aqueous solution of NaHCO3, water and methanol (3 x 15 cm 3 ) and dried at 60°C in an oven overnight (yield: 1.21 g, 64% based on 4). 1

Preparation of methanetetrayltetrakis(benzene-4,1-diyl)tetrakis(aza))tetrakis (methan-1-yl-1-yliden)tetrabenzoic acid (H4MTA) (7)

The last synthesic step in H4MTA preparation was deesterification reaction of its ethyl ester [START_REF] Wang | Metalorganic frameworks for stimuli-responsive drug delivery[END_REF].

6 (270 mg, 0.26 mmol) was suspended and stirred in 150 cm 3 solution of 1 M NaOH in methanol and heated at 80°C for 2 h. After this time the reaction mixture was cooled down to ambient temperature, filtrated and the solvent was removed under vacuum. The crude product was dissolved in water and solution was subsequently acidified with concentrated HCl until pH = 1. Orange solid of 7 was precipitated, filtered off, washed with water and dried in an over at 50°C overnight (yield: 226 mg, 94% based on 6). 1

Synthesis of UPJS-13 and UPJS-14

A mixture of H4MTA (10 mg, 0.011 mmol) and Cd(NO3)2•4H2O (27 mg, 0.088 mmol)

or Zn(NO3)2•6H2O (6.5 mg, 0.022mmol) were dissolved in a solvent mixture of DMF/H2O (6 cm 3 /0.5 cm 3 ) in a screwcapped vial. The vial was capped and placed in an oven and reaction mixtures were heated at 80°C for five days. Prepared orange crystals (see Fig. and after filtration process and 4x zoom.

Preparation of the samples for gas adsorption measurements a) As-synthesized samples (AS)

The as-synthesized samples for nitrogen adsorption experiments were prepared by repeating the synthetic procedure described in section 2.1.3 Synthesis of UPJS-13 and UPJS-14. After collecting of sufficient amount of the sample, the material was stored in the mother liquor. The collected material was further activated under vacuum at selected temperatures described in section 2.2.6 Nitrogen adsorption measurements.

b) Ethanol exchanged samples (EX)

The ethanol exchanged samples were prepared by immersing of the as-synthesized materials in ethanol for 14 days, while ethanol was changed every day. Ethanol exchanged samples were activated under vacuum at selected temperatures described in section 2.2.6

Nitrogen adsorption measurements.

c) Freeze-dried samples (FD)

The freeze-dried samples were prepared by immersing the as-synthesized materials in benzene for 14 days, while benzene was changed every day. Subsequently, the suspension of the sample in benzene was frozen at -20°C. After five freeze-thaw cycles, the benzene exchanged material in sample cells were placed under vacuum using a turbomolecular pump at -20°C for 24 h, and further heated under vacuum at selected temperatures described in section 2.2.6 Nitrogen adsorption measurements. Schematic representation of the described freezedrying process is shown in Fig. 3.

Fig. 3 Scheme of the freeze-drying process.

Methods and characterisation

Elemental analysis and ICP-MS

The elemental analysis was performed using a CHNOS Elemental Analyzer Vario MICRO from Elementar Analysensysteme GmbH with a sample weight of approximately 3 mg. The determination of metal ions amount in as-synthesized aMOFs was carried out on the ICP-MS 7700 instrument developed by Agilent Technologies, in the atmosphere of argon.

Before measurements, the samples were mineralised in hot aqua regia.

Infrared spectroscopy

The infrared spectra of the samples were measured at ambient temperature and recorded using an Avatar FTIR 6700 spectrometer in the wavenumber range of 4000-400 cm -1 with 64 repetitions for a single spectrum. Materials were measured using KBr technique in mass ratio 1: 100 (sample: KBr). Potassium bromide was before measurements dried in an oven at 500°C for 4 h and cooled in a desiccator.

NMR spectroscopy

Nuclear magnetic resonance data were collected on a Varian VNMRS 600 spectrometer operating at 599.87 MHz for 1 H and 150.84 MHz for 13

Thermal analysis

The thermal behaviour of prepared samples was studied by thermogravimetric analysis (TGA) with a sample weight of approximately 10 mg, using platinum crucibles. Samples were heated in the temperature range of 30-800°C with a heating rate of 10°C.min -1 in a dynamic air atmosphere with a flow rate of 50 cm 3 .min -1 , using TGA Q600 apparatus.

X-ray diffraction experiments

Single-crystal diffraction analysis were collected on a Bruker D8 VENTURE Kappa Duo PHOTON 100 diffractometer using MoKα radiation (λ = 0.71073 Å). The sample crystals were cooled to -150°C by using an Oxford Cryostream cooler. Experimental data were processed by the diffractometer software. Powder X-ray diffraction (PXRD) experiments were done in reflection geometry using a Bruker D8 ADVANCE ECO multipurpose diffractometer.

In order to achieve a parallel and clean X-ray beam for PXRD experiments, initially divergent Cu-Kα radiation (λ = 1.54056 Å) emitted from copper X-ray lamp was further guided through the multi-layered mirror and set of slits. Powder samples were loaded in a glass frame and PXRD experiments were done by 2θ continuous scan at 0.5°.min -1 from 10° to 100° so that scattered photons were recorded every 0.02° using a NaI scintillation detector at ambient temperature.

Gas adsorption measurements

Nitrogen adsorption isotherms were taken using a Quantachrome Nova 1200e apparatus at -196°C. Prior to nitrogen adsorption, the samples were outgassed at different temperatures (60, 80, 100, 120, 150 and 200°C) for 12 h under the vacuum to remove solvent molecules from the pores. The adsorption isotherms of desolvated samples were collected in a relative pressure range from p/p0 = 0.005 to 0.95. Based on the nitrogen adsorption measurements, the BET specific surface area (SBET) of each sample was evaluated using adsorption data in a p/p0 range from 0.05 to 0.20.

The gas adsorption measurements at 30°C and pressures up to 20 bar were made with carbon dioxide and methane using a homemade high-throughput instrument [START_REF] Wiersum | Experimental screening of porous materials for high pressure gas adsorption and evaluation in gas separations: Application to MOFs (MIL-100 and CAU-10)[END_REF]. Gas adsorption was measured via a manometric gas dosing system on both samples in parallel. The amounts of gas adsorbed were calculated by an equation of state using the Reference Fluid Thermodynamic and Transport Properties (REFPROP) software package 8.0 of the National Institute of Standards and Technology (NIST) [START_REF] Lemmon | MO reference fluid thermodynamic and transport properties[END_REF]. Approximately 100 mg of sample was used, and the sample was thermally activated individually in-situ under a primary vacuum, at a given final temperature overnight. Methane was of 99.9995% purity (N55), carbon dioxide was of 99.995% purity (N45) and gases were obtained from Air Liquide. The weight of the samples used in the high-pressure adsorption experiments was approximately 100 mg and before measurements, the freeze-dried samples were activated at 80°C under vacuum for 12h.

Results and discussion

Synthesis and NMR spectroscopy

The H4MTA ligand used in the preparation of aMOFs in the present study was synthesized by the seven-step organic synthesis (see Fig. 1). First, a tetraphenylmethane core was synthesized by the condensation reaction of triphenylmethanol and aniline and the prepared product was subsequently deaminated. Further reactions led to the introduction of nitro groups using fuming acid, which were subsequently reduced to amine groups. Described reactions led to the preparation of the first essential component required for the construction of H4MTA. The second basic component, ethyl 4-nitrosobenzoate, was prepared by transforming the nitro group to the nitroso group using oxone. The resulting H4MTA acid was prepared by the condensation reaction of ethyl 4-nitrosobenzoate with tetrakis-(p-aminophenyl)methane in anhydrous methanol and prepared ethyl ester was further deesterified in a concentrated solution of sodium hydroxide in methanol, followed by acidifying the solution to pH = 1.

NMR spectroscopy was used for molecular structure determination of all prepared organic compounds (see 2.1.2 Synthesis of H4MTA). The procedure to assign the NMR data of derivative 6 is described as an example. Compound 6 consists of twelve carbons. The carbonyl carbon is easily detectable in 13 The successful synthesis of the resulting tetrahedral tetraazo-tetracarboxylic acid was confirmed by 1 H and 13 C NMR measurements and corresponding spectra with assignments of hydrogen and carbon atoms are depicted in Fig. 4 and 2D NMR 1 H, 13 C-HSQC and 1 H, 13 H4MTA was used for the preparation of UPJS-13 (AS) and UPJS-14 (AS) using zinc(II)

and cadmium(II) nitrates in DMF/H2O at 80°C. The solvent molecules, DMF and water located in the cavities of the prepared compounds were exchanged with ethanol (UPJS-13 (EX) and

UPJS-14 (EX)).

A third variant of the materials was prepared by the freeze-drying process using benzene (UPJS-13 (FD) and (UPJS-14 (FD)). All described aMOFs materials were subsequently studied by nitrogen adsorption to find the best textural properties and after finding the best activation conditions, the selected compounds were tested as adsorbents for greenhouse gases, carbon dioxide and methane, by high-pressure adsorption measurements.

Single crystal and powder X-ray diffraction experiments

Since the materials were prepared in the form of crystals (see Fig. 2), compounds were first subjected to single-crystal X-ray diffraction analysis to solve their crystal structures.

Because the samples did not provide a diffraction image, even after selecting other crystals, the materials were subjected to powder X-ray diffraction (PXRD) experiments. These

Infrared spectroscopy

The infrared spectra of as-synthesized, ethanol exchanged and freeze-dried samples are shown in Fig. 6 and the assignment of absorption bands is summarised in Table 1. The IR spectra of both as-synthesized samples (see Fig. 6, black lines) exhibit broad band centred at about 3400 cm -1 which can be attributed to the OH stretching vibrations (ν(OH)) of water molecules, used in the synthesis. The presence of MTA linker in the prepared materials is evident from the intensive adsorption bands at 1603 cm -1 and 1378 cm -1 which can be assigned to antisymmetric and symmetric stretching vibrations of coordinated carboxylate groups (νas(COO -) and vs(COO -)). The presence of MTA in prepared materials demonstrated absorption band at 1563 cm -1 for UPJS-13 (AS) and 1543 cm -1 for UPJS-14 (AS) which can be attributed to the N=N stretching vibrations of an azo bond. The stretching vibration of aromatic CH (ν(CH)ar) groups of MTA was found at 3053 cm -1 for UPJS-13 (AS) and 3047 cm -1 for UPJS-14 (AS). The stretching vibrations of aliphatic CH groups (ν(CH)aliph) of DMF molecules adsorbed in the porous system of samples were observed in the region under 3000 cm -1 (2926 and 2853 cm -1 for UPJS-14 (AS); 2929 and 2850 cm -1 for UPJS-14 (AS)). The presence of DMF is also evident from the strong absorption band of carbonyl group ν(C=O) located at 1656 cm -1 and the ν(C-N) vibration at 1253 cm -1 for both compounds. In the IR spectra of ethanol exchanged samples (see Fig.

Thermal stability study

The thermal behaviour and stability of as-synthesized, ethanol exchanged and freezedried compounds were investigated using thermogravimetry and obtained TG curves are shown in Fig. 7. The AS samples (see black curves in Fig. 7) are thermally stable after heating to 80°C, above this temperature in the temperature range 80-380°C desolvation processes take place in two overlapping decomposition steps. In mentioned range total mass loss of 21.6 wt. % for

UPJS

Gas adsorption

Nitrogen adsorption measurements

To investigate the porosity of prepared aMOFs, nitrogen adsorption measurements at -196°C were performed. The adsorption measurements were realised on as-synthesized (AS), ethanol exchanged (EX) and freeze-dried (FD) samples. Ethanol exchanged samples were prepared by immersing of as-synthesized materials in ethanol. Freeze-dried samples were prepared after replacing high boiling point solvent (DMF, b.p. 153.0°C) inside the channels of prepared materials for benzene (b.p. 80.1°C, m.p. 5.5°C). Samples were further frozen and benzene was removed under vacuum by sublimation. Samples prepared by the described procedure were thermally activated at different temperatures (60, 80, 100, 120, 150 and 200°C) under vacuum for 12h. Obtained adsorption isotherms are presented in Fig. 8 and calculated BET surface areas (SBET) are summarised in Table 2 and shown in Fig. 9. The nitrogen adsorption measurements revealed a type I isotherm according to IUPAC classification, typical for microporous materials. The small difference in the shape of measured and ideal isotherm could be explained by the presence of weaker attractive forces between nitrogen molecules with pore walls or by the nitrogen interaction with a variety of active surface sites and other vacant sites formed during the activation procedures (dislocation, defects, electron-rich azo group).

For as-synthesized materials containing DMF/water molecules in the pores, it is characteristic that as the activation temperature increases, the surface area gradually increases up to 100°C (see black curves in Fig. 9a). Above this temperature, there is no more pronounced increase in SBET and the values are almost constant. This is caused by the gradual removal of solvent molecules from the pore system of materials. The calculated surface area at 100°C were 415 m 2 .g -1 for UPJS-13 (AS) and 629 m 2 .g -1 for UPJS-14 (AS). After activating samples at 200°C, a slight decrease in surface area was observed (see Fig. 8, Table 2).

For UPJS-13 (EX), the trend in surface area values at selected activation temperature is different. The maximum SBET of 721 m 2 .g -1 was reached at 80°C and by subsequent heating at 100°C decreases to the value of 210 m 2 .g -1 . The further increase in temperature does not have a significant effect on the textural properties (see red curves in Fig. 9), probably due to the collapse of the polymer framework. Because ethanol has a low boiling point, its release at 100°C occurs all at once, which lead to pore perforation associated with the destruction of the framework. The sample UPJS-14 (EX) showed similar behaviour compared to UPJS-14 (AS)

and gradually increased the SBET with increasing temperature up to 80°C and the maximal surface area after activation at 100°C was 880 m 2 .g -1 .

The best textural properties were observed for materials after the freeze-drying process (see green curves in Fig. 9). The highest values of surface areas were calculated after activation of the samples at 80°C (see green curves in Fig. 9, Table 2) and corresponding values were 830 m 2 .g -1 (pore volume, Vp = 0.57 cm 3 .g -1 ; pore diameter, d = 0.73 nm calculated by DFT) for UPJS-13 (FD) and 1057 m 2 .g -1 (pore volume, Vp = 0.72 cm 3 .g -1 ; pore diameter, d = 0.87 nm calculated by DFT) for UPJS-14 (FD). Gradual heating to a 120°C leads to a slight decrease in SBET values and further heating did not affect the values of surface area for both compounds.

It is to note, that the observed SBET values for UPJS-13 and UPJS-14 are higher compared with MOFs constructed from shorter tetrahedral linker, methanetetrabenzoate (MTB)

containing Ca(II) (126 m 2 .g -1 ) [START_REF] Almáši | A series of four novel alkaline earth metal-organic frameworks constructed of Ca(II), Sr(II), Ba(II) ions and tetrahedral MTB linker: Structural diversity, stability study and low/high-pressure gas adsorption properties[END_REF], Zn(II) (248 m 2 .g -1 ) [START_REF] Almáši | Synthesis, characterisation and sorption properties of zinc(II) metal-organic framework containing methanetetrabenzoate ligand[END_REF], Co(II) (356 m 2 .g -1 ) [START_REF] Cheon | Selective gas adsorption in a microporous metal-organic framework constructed of Co(II)4 clusters[END_REF], Ba(II) (358 m 2 .g -1 ) [START_REF] Almáši | A series of four novel alkaline earth metal-organic frameworks constructed of Ca(II), Sr(II), Ba(II) ions and tetrahedral MTB linker: Structural diversity, stability study and low/high-pressure gas adsorption properties[END_REF], Cu(II) (526 m 2 .g -1 ) [START_REF] Ma | Freeze drying significantly increases permanent porosity and hydrogen uptake in 4,4-connected metal-organic frameworks[END_REF] and Ni(II) (700 m 2 .g -1 ) [START_REF] Almáši | A novel nickel metal-organic framework with fluorite-like structure: Gas adsorption properties and catalytic activity in Knoevenagel condensation[END_REF] ions. A further comparison could be performed with a copper(II) MOF [Cu2(MTC)(H2O)2]•14DMF•5H2O [START_REF] Almáši | A series of four novel alkaline earth metal-organic frameworks constructed of Ca(II), Sr(II), Ba(II) ions and tetrahedral MTB linker: Structural diversity, stability study and low/high-pressure gas adsorption properties[END_REF], which contains similar extended tetrahedral linker MTC (methanetetra(biphenyl-pcarboxylate) to MTA described in the present study. The methanol exchanged and vacuum dried compounds, both activated at 60°C exhibited surface area of 791 m 2 .g -1 and its freeze-dried form 1020 m 2 .g -1 . The zinc(II) analogue with formulae [Zn2(MTC)(H2O)2] may also be mentioned, which exhibited after dichloromethane exchange a surface area of 1170 m 2 .g -1 [START_REF] Weng | Solvent-induced single-crystal to single-crystal transformation of a 2D coordination network to a 3D metal-organic framework greatly enhances porosity and hydrogen uptake[END_REF] and calculated SBET is similar to UPJS-14 (FD). The highest reported surface area was published for zirconium(IV) MOF containing MTC linker with a composition of [Zr6(OH)16(MTC)2] and SBET = 3411 m 2 .g -1 [START_REF] Zhang | Symmetry-guided synthesis of highly porous metal-organic frameworks with fluorite topology[END_REF][START_REF] Wang | Molecular vise approach to create metalbinding sites in MOFs and detection of biomarkers[END_REF].

In summary, it can be concluded that UPJS-14 material showed better textural properties compared to UPJS-13, regardless of the post-synthetic treatment and activation conditions. The highest values of surface areas of prepared materials were observed for samples after the freeze-drying process, followed by ethanol-exchanged and as-synthesized samples.

Compound UPJS-14 (FD) activated at 80°C exhibited large surface area compared with other reported MOF compounds containing similar extended tetrahedral linker.

Table 2 The summary of calculated specific surface areas (SBET) for UPJS-13 and UPJS-14

after activation process at different temperatures.

Sample Activation temperature (°C)

SBET surface area (m 2 .g -1 )

As

High-pressure carbon dioxide and methane adsorption measurements

As major components of greenhouse gases, carbon dioxide and methane have also been studied as probe molecules to investigate the sorption properties of prepared aMOFs. CO2 and CH4 high-pressure adsorption measurements were performed at 30°C and pressure up to 20 bar on freeze-dried samples activated at 80°C. Measured adsorption isotherms are shown in Fig. 10 and obtained maximal adsorption capacities at 1 and 20 bar are summarised in Table 3 -78°C for CO2) and described differences may influence the maximal adsorption uptake of gases.

aMOFs storage capacities of carbon dioxide at 1 bar and 30°C were 0.94 mmol.g -1 (4.14 wt. %) for UPJS-13 FD and 0.8 mmol.g -1 (3.52 wt. %) for UPJS-14 FD. With increasing pressure, an increase in the amount of adsorbed carbon dioxide was observed. The maximal adsorption uptake and corresponding adsorption capacities were 6.82 mmol.g -1 (30.01 wt. %)

for UPJS-13 FD and 5.58 mmol.g -1 (24.56 wt. %) for UPJS-14 FD at 30°C and 20 bar. Both compounds show high CO2 storage capacities, however, the UPJS-13 FD framework showed a higher affinity for CO2 compared to UPJS-14 FD, although it has a smaller surface area. Due to the presence of an electron-deficient carbon atom, carbon dioxide is expected to interact with protic electronegative functional groups, leading to strong chemisorption. This simple interaction has so far led to a number of nanoporous polymers in which nitrogen-rich functions are incorporated, such as triazine [START_REF] Das | Unprecedented high temperature CO2 selectivity and effective chemical fixation by a copper-based undulated metal-organic framework[END_REF][START_REF] Mukhtar | Synthesis-structure-property relationship of nitrogen-doped porous covalent triazine frameworks for pre-combustion CO2 capture[END_REF], tetrazole [START_REF] Zhang | Tetrazole-based porous metal-organic frameworks for selective CO2 adsorption and isomerisation studies[END_REF], imidazole [START_REF] Dhankhar | Construction of 3D lanthanide based MOFs with pores decorated with basic imidazole groups for selective capture and chemical fixation of CO2[END_REF][START_REF] Webb | The significance of metal coordination in imidazole-functionalised metal-organic frameworks for carbon dioxide utilisation[END_REF], imide [START_REF] Wang | Constructing redox-active microporous hydrogen-bonded organic framework by imidefunctionalisation: Photochromism, electrochromism, and selective adsorption of C2H2 over CO2[END_REF] and amines [START_REF] Kim | Cooperative carbon capture and steam regeneration with tetraamine-appended metal-organic frameworks[END_REF][START_REF] Salehi | Improving CO2/CH4 and CO2/N2 adsorptive selectivity of Cu-BTC and MOF-derived nanoporous carbon by modification with nitrogen-containing groups[END_REF]. Increased CO2 adsorption capacity in UPJS-13 (FD) and UPJS-14 (FD) can be explained by an interaction between CO2 molecules and adsorption sites represented by a lone pair on the azo functional groups located in the molecular structure of the MTA linker. In the case of azo group, the interaction with CO2 was also confirmed by computational studies, in which the relative binding affinity of CO2 to the -N=Ngroup using trans-azobenzene as a model system were performed. Calculations have shown that the lowest energy structure of the CO2 / trans-azobenzene complex indicates that carbon dioxide preferentially binds to the azo group of trans-azobenzene by physisorption with a binding energy of 17.0 kJ.mol -1 . CO2 also has a favourable interaction with the aromatic ring, including a large stabilizing energy, 14.4 kJ.mol -1 [START_REF] Patel | Unprecedented high-temperature CO2 selectivity in N2-phobic nanoporous covalent organic polymers[END_REF]. It is known that MOFs with coordinatively unsaturated sites (CUSs) offer interesting possibilities for tuning the affinity of these materials towards certain adsorbates, potentially increasing their selectivity and storage capacity [START_REF] Kökçam-Demir | Coordinatively unsaturated metal sites (open metal sites) in metal-organic frameworks: Design and applications[END_REF]. According to the literature data, the high-pressure CO2 adsorption measurements were performed for two compounds containing MTB linker. Nickel(II) MOF (SBET = 700 m 2 .g -1 ) adsorbed 18.05 wt. % (4.1 mmol.g -1 ) of CO2 [START_REF] Almáši | A novel nickel metal-organic framework with fluorite-like structure: Gas adsorption properties and catalytic activity in Knoevenagel condensation[END_REF] and lead(II) compound (SBET = 980 m 2 .g -1 )

19.81 wt. % (4.5 mmol.g -1 ) of CO2 [START_REF] Almáši | Microporous lead-organic framework for selective CO2 adsorption and heterogeneous catalysis[END_REF][START_REF] Almáši | Efficient and reusable Pb(II) metalorganic framework for Knoevenagel condensation[END_REF] Methane is the main component of natural gas and the second most abundant greenhouse gas in Earth´s atmosphere. CH4 is a non-polar molecule with zero quadrupole moment and has a low volumetric energy density, which poses a challenge for efficient storage to reduce its concentration in the atmosphere or for energy application. For this reason, methane was also used as an adsorbate for high-pressure adsorption and measured isotherms are presented in Fig. 10. Materials adsorb methane with a maximal storage capacity of 0.22 mmol.g -1 (0.35 wt. %)

for UPJS-13 (FD) and 0.36 mmol.g -1 (0.58 wt. %) for UPJS-14 (FD) at 30°C and 1 bar and 3.02 mmol.g -1 (4.84 wt. %) for UPJS-13 (FD) and 3.98 mmol.g -1 (6.38 wt. %) for UPJS-14

(FD) at 30°C and 20 bar. It could be noted that the adsorbed amounts are approximately four/five times lower compared to carbon dioxide. Contrary, the values of stored CH4 are relatively high and can be explained by the pore size of aMOF materials. Several theoretical studies have shown that the optimum pore size for methane storage is 8 Å, which corresponds to distance between pore walls of about twice larger, than the molecule diameter [START_REF] Alcaniz-Monge | Methane storage in activated carbon fibres[END_REF][START_REF] Getman | Review and analysis of molecular simulations of methane, hydrogen, and acetylene storage in metal-organic frameworks[END_REF]. As it was described above, the effective pore size of UPJS-13 (FD) and UPJS-14 (FD) based on nitrogen adsorption are 7.3 and 8.7 Å; thus it could be an explanation for increased CH4 adsorption.

A comparison of the adsorption capacity with compounds containing shorter tetrahedral carboxylate linker (MTB) has shown that the CH4 adsorption capacities were reported for Ni(II) [START_REF] Almáši | A novel nickel metal-organic framework with fluorite-like structure: Gas adsorption properties and catalytic activity in Knoevenagel condensation[END_REF] and Pb(II) [START_REF] Almáši | Microporous lead-organic framework for selective CO2 adsorption and heterogeneous catalysis[END_REF] compounds with a methane storage capacity of 3.29 wt. % (2.05 mmol.g - 1 ) and 3.05 wt. % (1.9 mmol.g -1 ) at 30°C and 21 bar, respectively. The highest documented CH4 product, Basolite A520 (aluminum fumarate, SBET = 1300 m 2 .g -1 ) [START_REF] Gaab | The progression of Al-based metal-organic frameworks -From academic research to industrial production and applications[END_REF] have also shown promise as methane sorbents with methane storage capacity of 6.5 mmol.g -1 (10.43 wt. %) at 25°C and 20 bar and 3.80 mmol.g -1 (6.10 wt. %) at room temperature and pressure 20 bar, respectively.

Conclusion

The present study dealt with the preparation, characterisation and application of two novel aMOF compounds as adsorbents of greenhouse gases, carbon dioxide and methane. In the first phase, the extended nitrogen-rich tetraazo-tetracarboxylic acid, H4MTB was prepared by a seven-step organic synthesis. Spectroscopic and NMR measurements confirmed its successful preparation. The next step was the preparation of aMOF compounds in combination with H4MTB and zinc(II) or cadmium(II) cations under formation of as-synthesized materials, UPJS-13 (AS) and UPJS-14 (AS). In order to facilitate the activation of the compounds, the solvent molecules located in the cavities of the as-synthesized materials were exchanged with ethanol (UPJS-13 (EX) and UPJS-14 (EX)). Another variant of the compounds was prepared by a freeze-drying process using benzene (UPJS-13 (FD) and UPJS-14 (FD)). The compounds were characterised by elemental analysis, infrared spectroscopy and PXRD. All prepared aMOFs were studied by nitrogen adsorption to find the best activation conditions. The compounds showed the highest surface areas after the freeze-drying process and at an activation temperature of 80°C (SBET = 830 m 2 .g -1 for UPJS-13 (EX) and SBET = 1057 m 2 .g -1 for UPJS-14 (EX)). aMOF materials were subsequently tested as greenhouse gases adsorbents, carbon dioxide and methane, by high-pressure adsorption measurements at 30°C and pressure up to 20 bar. The maximal gas storage capacities were 30.01 wt. % for UPJS-13 FD, 24.56 wt. % for UPJS-14 FD of CO2 and 4.84 wt. % for UPJS-13 FD and 6.38 wt. % for UPJS-14 FD of CH4

  dioxide is a major by-product of fossil fuel combustion, electricity generation and other anthropogenic activities. Major advances in carbon capture technology could provide power plants and road transport with an efficient and inexpensive way to remove carbon dioxide from their flue gas emissions, which is necessary to reduce greenhouse gas emissions to slow global warming and climate change. The 2030 Climate and Energy Framework commits all EU member states to a 40% reduction in greenhouse gas emissions over the coming years[33]. For this reason, it is necessary to develop new and effective carbon dioxide and methane adsorbents as the main greenhouse gases. Many porous materials such as carbon dioxide adsorbents, e.g. MOFs or porous oxides, are modified with nitrogen-containing amine molecules to capture the CO2 (chemisorption) at low-temperature steam to flush out the CO2 for other uses or to sequester it underground are currently being developed and intensively studied. Examples are tetraamine-appended MOF Mg2(dobpdc) (dobpdc = 4,4′-dioxidobiphenyl-3,3′-dicarboxylate,

  (w); ν(C=O) 1709 (s); ν(C=C)ar. 1597 (w); ν(N=N) 1521 (w); δ(CCH)ar. 1085 (m), 1009 (m); γ(CCH)ar. 858 (m), 697 (s) cm -1 . 2D NMR experiments: 1 H, 13 C-HSQC and 1 H, 13 C-HMBC spectra of 7 are presented in Figs. S5 and S6 in ESI.

Fig. 1

 1 Fig. 1 Synthesis of methanetetrayltetrakis(benzene-4,1-diyl)tetrakis(aza))tetrakis(methan-1yl-1-yliden)tetrabenzoic acid (H4MTA) with corresponding synthetic conditions.

Fig. 2

 2 Fig. 2 Prepared orange crystals of a) UPJS-13 (AS) and b) UPJS-14 (AS) in screwcapped vials

  C. The mass concentration of all samples was approximately 10 mg/0.6 mL of DMSO-d6 or CDCl3, and the chemical shifts were referenced to the residual solvent peak ( 1 H NMR 2.50 ppm and 13 C NMR 39.5 ppm for DMSO-d6; 1 H NMR 7.26 ppm and 13 C NMR 77.0 ppm for CDCl3). The NMR data were recorded at 300 K, with chemical shifts () reported in parts per million and coupling constants (J) in Hertz. The 2D experiments gCOSY, gHSQC and gHMBC were run using the standard Varian software. All data were analysed using MNova 7.1.1 (2012) software. The spectral acquisition parameters are summarised in section 2.1.2 Synthesis of H4MTA and selected spectra are shown in Fig. 4 and Figs. S1-S7 in ESI.

Fig. 4 a

 4 Fig. 4 a) 1 H (600 MHz) and b) 13 C (151 MHz) NMR spectra of H4MTA in DMSO-d6 with assignments of hydrogen and carbon atoms.

  measurements aimed to confirm the amorphous composition of the whole bulk of prepared materials and confirm their amorphous character. Measured PXRD patterns of materials are shown in Fig. 5 and confirmed their disordered character. Aperiodic arrangements of atoms result in their PXRD patterns being dominated by broad "humps" caused by diffuse scattering and thus they are mostly indistinguishable from one another.

Fig. 5

 5 Fig. 5 Powder X-ray diffraction patterns of UPJS-13 (AS) and UPJS-14 (AS).

6 ,

 6 red lines) absorption bands belonging to the MTA linker are still present, but the characteristic vibration ν(C=O) around 1650 cm -1 for DMF molecules is no longer present. Described observation confirms the successful exchange of DMF molecules for ethanol. Ethanol molecules in the IR spectra showed a shift in ν(OH) vibration to lower wavelength values (3277 cm -1 for UPJS-13 (EX) and 3260 cm -1 for UPJS-14 (EX)) and also new aliphatic ν(CH)aliph vibrations appeared under 2930 cm -1 . In the IR spectra of freeze-dried compounds (see Fig.6, blue lines), no characteristic absorption bands originated from DMF or ethanol are present, which confirm the successful activation of UPJS-13 and UPJS-14 materials.

Fig. 6

 6 Fig. 6 Infrared spectra of as-synthesized (AS), ethanol exchanged (EX) and freeze-dried (FD) materials of a) UPJS-13 and b) UPJS-14.

- 13 (

 13 AS) and 32.0 wt. % for UPJS-14 (AS) was observed and correspond to release of solvent molecules (3 × DMF, 4 × H2O, clcd. mass loss 21.9 wt. % for UPJS-13 (AS); 4 × DMF, 5 × H2O, clcd. mass loss 31.8 wt. % for UPJS-14 (AS)). Further heating led to the decomposition of frameworks, which occurs in 380-530°C. Final thermal decomposition products were ZnO (observed residual mass 12.6 wt. %, clcd. residual mass 12.2 wt. % for UPJS-13 (AS)) and CdO (observed residual mass 17.4 wt. %, clcd. residual mass 16.9 wt. % for UPJS-14 (AS)). The desolvation process in ethanol exchanged samples takes place at a lower temperature (see red curves in Fig.7), the release of ethanol molecules starts at the beginning of heating and complete desolvation occurs about 180°C. The desolvated forms of UPJS-13 (EX) and UPJS-14 (EX) are thermally stable from 180-420°C as is seen from the plateau on TG curves. The decomposition of polymeric frameworks takes place in two overlapping decomposition steps, similarly to the as-synthesized samples. As can be seen from obtained TG curves for EX materials, the solvent exchange process significantly impacts the compounds' activation temperature. The TG curves of the freeze-dried samples (see green curves in Fig.7) confirm the successful activation of the compounds, as no weight loss is observed in the temperature range 30-420°C. The decomposition of UPJS-13 (FD) and UPJS-14 (FD) frameworks, [Zn2(MTA)] and [Cd2(MTA)], takes place in temperature range 420-530°C with residual masses 15.5 wt. % and 33.9 wt. % corresponding to the formation of ZnO and CdO as final decomposition products (clcd. residual masses 15.7 wt. % for ZnO and 34.4 wt. % for CdO).

Fig. 7

 7 Fig. 7 Thermogravimetric curves of as-synthesized, ethanol exchanged and freeze-dried samples of a) UPJS-13 and b) UPJS-14 measured in an air atmosphere and temperature range of 30-800°C.

Fig. 8

 8 Fig. 8 Nitrogen adsorption isotherms of a) as-synthesized, b) ethanol exchanged and c) freezedried samples of UPJS-13 and UPJS-14 after activation at 60, 80, 100, 120, 150 and 200°C.

Fig. 9

 9 Fig. 9 Calculated BET surface areas from nitrogen isotherms of activated materials at 60, 80, 100, 120, 150 and 200°C for a) UPJS-13 and b) UPJS-14.

  capacities were reported for NU-125 (Cu(BTTI), BTTI = 5,5´,5´´-(4,4´,4´´-(benzene-1,3,5triyl)tris(1H1,2,3-triazole-4,1-diyl))triisophthalate, SBET = 3120 m 2 .g -1 ) [68] which can store 11.47 mmol.g -1 (18.40 wt. %) at 25°C and 20 bar and BUT-22 (Al3(O)(OH)(H2O)2(PPTTA)3/2, PPTTA = (1,4-phenylenebis(pyridine-4,2,6-triyl))tetrabenzoate, SBET = 4380 m 2 .g -1 ) [69] with storage capacity of 9.9 mmol.g -1 (15.88 wt. %) at 23°C and 20 bar. Other MOFs, such as [Co(BDP)] (BDP = 1,4-benzenedipyrazolate, SLANG = 2911 m 2 .g -1 ) [70] and commercial

Fig. 10

 10 Fig. 10 Methane and carbon dioxide high-pressure adsorption isotherms of UPJS-13 and UPJS-14 measured at 30°C and pressure up to 20 bar.

  Figures:

  Fig. S1 1 H (600 MHz, DMSO-d6) and 13 C (151 MHz, DMSO-d6) NMR spectra of the derivative 6.

Fig. S2 1 H

 1 Fig. S2 1 H, 1 H-COSY spectrum (600/600 MHz, DMSO-d6) of the derivative 6.

Fig. S4 1 H

 1 Fig. S4 1 H, 13 C-HMBC spectrum (600/151 MHz, DMSO-d6) of the derivative 6.

Fig. S5 1 H

 1 Fig. S5 1 H, 13 C-HSQC spectrum (600/151 MHz, DMSO-d6) of the derivative 7.

2.1.2.3 Synthesis of tetrakis(p-nitrophenyl)methane (3)

	MHz, CDCl3) δ (ppm): 67.0; 125.8; 127.4; 131.1; 146.7 ppm. Elemental analysis for C25H20
	(320.43 g.mol -1 ) calculated: C, 93.71%; H, 6.29%; measured: C, 92.67%; H, 6.19%. IR (KBr):
	ν(CH)ar. 3080 (w), 3052 (w), 3029 (w), 3011 (w); ν(C=C)ar. 1591 (w), 1490 (m); δ(CCH)ar. 1181
	(w), 1080 (w), 1034 (w); γ(CCH)ar. 748 (s), 699 (s) cm -1 .
	Tetrakis(p-nitrophenyl)methane was prepared by nitration reaction of 2 using fuming
	acid. Fuming acid (nitric acid, 99% HNO3, 12.81 cm 3 ; 0.3 mol) was cooled to ≈ -10°C using
	H2O/NaCl bath and 2 (2.56 g; 8 mmol) was added to the solution in small portions as the
	reaction is extremely exothermic. After complete addition of 2, acetic anhydride (4.27 cm 3 ; 45
	mmol) was added dropwise and the reaction mixture was stirred for 30 min. The last step was
	a dilution of the reaction mixture into glacial acetic acid (17.1 cm 3 ). A yellow precipitate of 3
	was filtered off, washed with acetic acid, methanol and dried in an oven at 50°C overnight
	(yield 1.63 g, 40.8% based on 2). Finally, the product was recrystallised from toluene, resulting
	in a reaction yield of 30%. 1 H NMR (600 MHz, DMSO): δ = 7.61 (8H, d, J = 9.01 Hz); 8.22
	(8H, d, J = 9.01 Hz) ppm. Elemental analysis for C25H16N4O8 (500.42 g.mol -1 ) calculated: C,
	60.00%; H, 3.22%; N,11.20%; N, 11.20%; measured C, 60.31%; H, 3.18%; N, 11.02%. IR
	(KBr): ν(CH)ar. 3109 (w), 3079 (w); ν(C=C)ar. 1604 (m), 1589 (m), 1493 (w); νas.(C-NO2) 1520
	(s); νs.(C-NO2) 1337 (s); δ(CCH)ar. 1106 (m), 1009 (w); γ(CCH)ar. 837 (s), 743 (s) cm -1 .
	2 was filtered off under reduced
	pressure, washed with DMF, methanol and dried in an oven at 70°C overnight (yield: 8.83 g,
	92.4% based on 1). 1 H NMR (600 MHz, CDCl3) δ (ppm): 7.21 (m, 20H) ppm. 13 C NMR (151

  C NMR spectrum because of the chemical shift 166.2 ppm. The

	carbon peak at 166.2 ppm showed long-range couplings to signals of proton at 4.42 ppm (q, J
	= 7.1 Hz), protons at 8.19 ppm (d, J = 8.8 Hz) and protons at 7.94 ppm (d, J = 8.8 Hz). The
	proton signal at 4.42 ppm was easily assigned as H-5 because of the splitting pattern as quartet
	and chemical shift of corresponding carbon peak at 61.4 ppm in the HSQC spectra. The
	chemical shift for H-6, methyl group, is shown as a triplet of splitting pattern at the lowest
	frequency at 1.43 ppm. The protons at 8.19 ppm were H-3'',5''. They correlated with 130.7 ppm
	of 13 C peak at HSQC spectrum and were coupled to both C-2'',6'' and C-1'' in the HMBC

spectrum. The chemical shift for H-2',6' (7.54 ppm (d, J = 8.8 Hz)) was assigned by HMBC correlation with quaternary carbon C-1 (65.6 ppm). Protons H-3',5' (7.92 ppm (d, J = 8.8 Hz))

were determined from the COSY experiment. The C-4' is long-range coupled to H-2',6'. The chemical shift for C-1' was assigned by HMBC correlation with H-3',5'.

Table 1

 1 Assignment of vibrations and corresponding wavenumbers to characteristic absorption bands in IR spectra of prepared and pretreated materials.

	ν(OH)	3440 (w, br)	3277 (w, br)	-	3407 (w, br)	3260 (w, br)	-
	ν(CH)ar	3053 (w)	3053 (w)	-	3047 (w)	3047 (w)	-
	ν(CH)aliph	2926 (w)	2926 (w)	-	2929 (w)	2929 (w)	-
		2853 (w)	2853 (w)		2850 (w)	2850 (w)
	ν(C=O)	1656 (s)	-	-	1656 (s)	-	-
	ν(N=N)	1563 (w)	1563 (w)	1563 (w)	1543 (w)	1543 (w)	1543 (w)
	νas(COO -)	1603 (s)	1603 (s)	1603 (s)	1603 (s)	1603 (s)	1603 (s)
	ν(CC)ar	1490 (m)	1490 (m)	1490 (m)	1493 (m)	1493 (m)	1493 (m)
	νas(COO -)	1378 (s)	1378 (s)	1378 (s)	1378 (s)	1378 (s)	1378 (s)
	ν(CN)	1253 (m)	-	-	1253 (m)	-	-
	δ(COO -)	785 (s)	785 (s)	785 (s)	787 (s)	787 (s)	787 (s)

wweak, mmedium, sstrong, brbroad, araromatic, aliphaliphatic, ssymmetric, asantisymmetric

UPJS-13 (AS) UPJS-13 (EX) UPJS-13 (FD) UPJS-14 (AS) UPJS-14 (EX) UPJS-14 (FD)

  . The absence of district plateaus in the isotherms indicated that the maximal capacities were still not achieved and compounds are not saturated by the gases at 20 bar. Both selected probes are non-

	polar molecules with different quadrupole moments (0 C.m 2 for CH4, 14.3×10 40 C.m 2 for CO2),
	kinetic diameters (3.8 Å for CH4, 3.3 Å for CO2) and different boiling points (-161°C for CH4,

Table 3

 3 Adsorbed amount (in mmol.g -1 and wt. %) of CO2 and CH4 at 30°C and pressure 1/20 bar in freeze-dried compounds after activation at 80°C.

			CO2					CH4
			pressure / bar			pressure / bar
		1		20		1		20
		mmol.g -1 wt. % mmol.g -1 wt. % mmol.g -1	wt. %	mmol.g -1 wt. %
	UPJS-13 (FD)	0.94	4.14	6.82	30.01	0.22	0.35	3.02	4.84
	UPJS-14 (FD)	0.80	3.52	5.58	24.56	0.36	0.58	3.98	6.38

Acknowledgements

This work was supported by the Scientific Grant Agency of the Slovak Republic (VEGA) project no. 1/0865/21 and from P. J. Safarik University project no. VVGS-2020-1667.

at 30°C and 20 bar. The increased adsorption capacity of carbon dioxide can be explained by the interaction of CO2 molecules with azo groups located in the structure of MTA ligand, or by interaction with CUSs. And the increased adsorption capacity of methane can be explained by the suitable pore size of the prepared materials.

Conflict of interest statement

No potential conflict of interest was reported by the authors.

SUPPLEMENTARY MATERIAL

Conflict of interest statement

No potential conflict of interest was reported by the authors.

Conflict of Interest


MD Marion Pinsard

PhD Nicolas Mouchet

MD Ludivine Dion

MD, PhD Thomas Bessede

MD, PhD Martin Bertrand

MD, PhD Emile Darai

Pascale Bellaud

MD Philippe Loget

MD, PhD Séverine Mazaud-Guittot

MD, PhD Xavier Morandi

MD, PhD Jean Leveque

MD, PhD Vincent Lavoué

MD Martha Duraes

Nyangoh Krystel
email: krystel.nyangoh.timoh@chu-rennes.fr

MD, PhD Timoh

MD Krystel Nyangoh Timoh

Anatomic and functional mapping of human uterine innervation

Keywords: Academic Research Unit Patients, None Interventions, None neuro-anatomy -uterine innervation -endometriosis -benign pelvic diseasechronic pelvic pain

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

INTRODUCTION

Complex benign pelvic diseases -such as chronic pelvic pain, endometriosis, myomas and adenomyosis-are frequent and can considerably affect a women quality of life [START_REF] Pynnä | The health-related quality of life of patients with a benign gynecological condition: a 2-year follow-up[END_REF]. Many studies suggest that they are associated with uterine and pelvic innervation abnormalities [START_REF] Anaf | Hyperalgesia, nerve infiltration and nerve growth factor expression in deep adenomyotic nodules, peritoneal and ovarian endometriosis[END_REF][START_REF] Quinn | Endometriosis: the consequence of uterine denervation-reinnervation[END_REF][START_REF] Quinn | Uterine innervation in fibroids: a qualitative study[END_REF][START_REF] Quinn | Differences in uterine innervation at hysterectomy[END_REF][START_REF] Tokushige | Different types of small nerve fibers in eutopic endometrium and myometrium in women with endometriosis[END_REF][START_REF] Tokushige | High density of small nerve fibres in the functional layer of the endometrium in women with endometriosis[END_REF][START_REF] Tokushige | Nerve fibres in peritoneal endometriosis[END_REF][START_REF] Zhang | Innervation of endometrium and myometrium in women with painful adenomyosis and uterine fibroids[END_REF]. Tokushigue et al demonstrated that patients with endometriosis have an autonomic innervation of the superficial layer of the endometrium unlike control patients [START_REF] Tokushige | High density of small nerve fibres in the functional layer of the endometrium in women with endometriosis[END_REF]. However, in a cohort of patients with myomas or adenomyosis, Zhang et al showed that only patients with pain had innervation of the functional layer as well as significantly higher innervation of the myometrium [START_REF] Zhang | Innervation of endometrium and myometrium in women with painful adenomyosis and uterine fibroids[END_REF]. Moreover, Ellet et al demonstrated that nerve fibers were present in the endometrium of patients with and without endometriosis in a cohort of patients with pain [START_REF] Ellett | Are endometrial nerve fibres unique to endometriosis? A prospective case-control study of endometrial biopsy as a diagnostic test for endometriosis in women with pelvic pain[END_REF]. Furthermore, Quinn et al described micro-neuromas within the myometrium in patients with chronic pelvic pain [START_REF] Quinn | Differences in uterine innervation at hysterectomy[END_REF]. These results suggest that uterine innervation is involved in complex benign pelvic diseases, and particularly in nociceptive mechanisms associated with pelvic pain. However, few data have been published about the organization and specificity of intrauterine innervation in the human and its relation to uterine portions and layers. New anatomical techniques such as immunohistochemistry, fluorochromes and computerassisted anatomical dissection (CAAD) have been developed to study pelvic neuro-anatomy in recent years, with clinical and surgical implications [START_REF] Timoh | Levator ani muscle innervation: Anatomical study in human fetus[END_REF][START_REF] Timoh | Detailed muscular structure and neural control anatomy of the levator ani muscle: a study based on female human fetuses[END_REF][START_REF] Alsaid | Tridimensional computer-assisted anatomic dissection of posterolateral prostatic neurovascular bundles[END_REF].

Our aim was to study the functional anatomy of uterine micro-innervation with a CAAD technique and to provide a comprehensive three-dimensional (3D) reconstruction to develop a clinical correlation for the practicing physician.

MATERIAL AND METHODS

Human fetuses

The fetal specimens were obtained from late miscarriages which were not secondary to chorioamnionitis or known genetic abnormalities, and without genito-urinary malformation.

All parents gave written consent for the scientific use of the cadaver. Only specimens without maceration, or morphological or macroscopic neurological abnormalities on pathology examination were used. The French Biomedicine Agency approved the study (PFS09-011).

The work was compliant with the provisions of the 2013 revised version of the Declaration of Helsinki. Four female fetuses, aged at 21, 24, 26, and 27 weeks of gestation, as determined from the crown-rump length(14), were studied. The external body aspect of each fetus was carefully restored after the autopsy.

Histological conditioning

The entire pelvis was removed en-bloc with the pelvic organ and pelvic bone, and was fixed in formalin (4% formaldehyde) for 6 days. Tissues were then cut into transverse or sagittal slices at 4-mm intervals. The tissue slices were placed in baskets, processed, and embedded in cardboard molds filled with paraffin. To avoid alteration of the visceral topographical relationship, the slices were kept warm in water at 37°C and then mounted whole on Superfrost glass slides. They were then dried at 56°C overnight. We obtained a total of 1000 to 2000 sections from each fetus, depending on the age and size of the specimen.

Staining and immunolabeling

The first section of each level was taken as the reference and was stained with Hematoxylin Eosin Safran (HES) to determine the main anatomical structures (collagen tissue in yellow, cytoplasm in pink, nuclei in purple).

Smooth muscles were detected with polyclonal antibodies against α-smooth muscle actin (SMA) [START_REF] Kobayashi | A Simple Method of Isolating Mouse Aortic Endothelial Cells[END_REF]. Neuronal markers were detected with polyclonal antibodies against protein S100 (PS100) for the labeling all nerves [START_REF] Hollabaugh | Neuroanatomy of the male rhabdosphincter[END_REF], peripheral myelin protein (PMP 22) for the somatic peripheral nerves [START_REF] Bremer | Axonal prion protein is required for peripheral myelin maintenance[END_REF], tyrosine hydroxylase (TH) for sympathetic nerves [START_REF] Sa | Pelvic Nerve Plexus Trauma at Radical and Simple Hysterectomy: A Quantitative Study of Nerve Types in the Uterine Supporting Ligaments[END_REF], vesicular acetylcholine transferase (VAChT) for parasympathetic neurons [START_REF] Usdin | Molecular biology of the vesicular transporter[END_REF], the neural isoform of nitric oxide synthase (nNOS) for the pro-erectile nerve bundles [START_REF] Moszkowicz | Neural Supply to the Clitoris: Immunohistochemical Study with Three-Dimensional Reconstruction of Cavernous Nerve, Spongious Nerve, and Dorsal Clitoris Nerve in Human Fetus[END_REF], vasointestinal peptide (VIP) for parasympathetic and sensitive nerve fibers [START_REF] Cohen | Neuropeptide Y and Calcitonin Gene-Related Peptide Modulate Voltage-Gated Ca2+ Channels in Mature Female Rat Paracervical Ganglion Neurons[END_REF], neuropeptide Y (NPY) for sympathetic and sensitive nerve fibers [START_REF] Cohen | Neuropeptide Y and Calcitonin Gene-Related Peptide Modulate Voltage-Gated Ca2+ Channels in Mature Female Rat Paracervical Ganglion Neurons[END_REF], calcitonin gene-related peptide (CGRP) for sensitive fibers [START_REF] Gnanamanickam | Innervation of the rat uterus at estrus: a study in full-thickness, immunoperoxidase-stained whole-mount preparations[END_REF], and oxytocin and relaxin-2 (RLN2) for nerves involved in contraction and relaxation of smooth muscle(23) (Table 1).

6

All immunostaining was automated (DISCOVERY Ultra system from Roche ®, Illkirch, France).The different steps were as follows: 1) Dewaxing (72°C) and rehydration (Discovery Wash -Ref: 07311079001-Roche); 2) Antigenic unmasking (citrate pH 6 or EDTA pH 8 at 95°C); 3) Inactivation of endogenous peroxidase with hydrogen peroxide; 4) Application and incubation of the primary antibody at the right dilution (Supplemental Table 1)

Image analysis

Two-dimensional (2D) serial stained and immunolabeled sections were used for the 3D reconstruction. Using high magnification (4× to 40×) analyses of the HES-stained sections, it was possible to identify the various anatomical structures (organs, bones, and fascia).

Subsequent sections, treated with an antibody against S100, were used to identify pelvic nerves and communicating branches. We compared the HES-stained sections with sections stained with specific antibodies against SMA and S100 firstly, and then with the other antibodies. In this way it was possible to study the path of the nerve fibers towards the uterus, and within its different parts and layers, and to determine the specificity of these nerves.

The computer system comprised a personal laptop computer (Windows XP) equipped with an Epson Perfection V750 digitization system, Silverfast AI digitization software (Ref B11B178071), Adobe Photoshop image-processing software, and Surfdriver software for Windows (Winsurf image reconstruction software, version 4.3). All sections were digitized at a resolution of 4800 dots per inch, and the images were then stacked and aligned. The brightness and contrast of the histological tissue images were adjusted using Adobe Photoshop. The pelvic anatomical structures and nerve fibers were outlined manually on all histological sections.

We also used HALO® software (version 3.0.311.255) to perform objective automated nerve quantification on the immunofluorescence slices (Figure 1) in three portions of the uterus (supra-vaginal cervix, intra-vaginal cervix, and uterine body), and in four uterine layers: peritoneal serosa, myometrium, the basal endometrial layer (deep layer adjacent to the myometrium which undergoes little menstrual change and is not expelled at the end of cycle), and the functional layer (containing the thick intermediate (spongy) layer, and the top thinner (compact) layer, undergoing significant menstrual changes and expelled at the end of cycle). The density of the nerve fibers was calculated as a percentage of the studied surface area.

Finally, a 3D analysis of the location, course, and distribution of the nerve fibers was carried out and an animated reconstruction generated (Supplemental Figure/video 1).

8

RESULTS

Uterine innervation in the human fetal models

Two-dimensional observations

The entry point of the uterine nerve fibers is lateral to the uterine isthmus and cervix. Nerve fibers arise directly from the inferior hypogastric plexus.

Organization of uterine innervation

Uterine innervation is centripetal and runs from the uterine serosa to the endometrium (Figure 1). The uterine nerves penetrate the myometrium and become less dense as they reach the endometrium.

Myometrial innervation is dense (Figure 1) and preferentially perivascular, but also sometimes peri-muscular (Figure 2). Uterine innervation formed a linear organization within the myometrium.

Endometrial innervation does exist but is sparse. We observed nerves within all the layers of the endometrium with a linear organization within the functional layer of the endometrium. (Supplemental Figure 2 and Supplemental Figure 3). Density of nerves in the uterus (Supplemental Figure 4)

Nerve density varies widely between the different portions of the uterus: denser at the cervix and less dense in the uterine body. Nerves penetrate the uterus at the uterine isthmus with most of them then clustering around the cervix and others running up and down.

Nerve density also varies between the different uterine layers as previously described according to the centripetal disposition of uterine innervation. It is denser at the periphery within the serosa particularly laterally to the cervix next to the parametrium and paracervix. Specificity / function of uterine innervation (Table 1)

We found four types of nerve labeling in the uterus: sympathetic (TH+ and NPY+), parasympathetic (VIP+), and sensitive nerve fibers (NPY+, CGRP1+ and VIP+). They are found in all portions and layers of the uterus (Supplemental Figure 5, Supplemental Figure 6, Supplemental Figure 7, Supplemental Figure 8).

The dominant nerve fibers are sympathetic (TH+). The second dominant nerve fibers are sympathetic and sensitive (NPY+). The third dominant nerve fibers are sensitive (CGRP1+) and the last are parasympathetic and sensitive (VIP+).

The preferential portions for these specific nerves are the cervix (TH+ with 79% of fibers, NPY+ with 73% of fibers and VIP+ 13%) and the uterine body for CGRP1+fibers (63% of nerves).

The preferential layers are the level of the serosa of the supra-vaginal cervix, i.e., in contact with the paracervix for TH+ fibers and the myometrium for NPY+ and CGRP+ fibers. TH+, NPY+, CGRP+ and VIP+ nerve fibers are present in the functional layer of the endometrium. NPY+ and CGRP+ fibers are the most common, and VIP+ the least; NPY+ and CGRP1+ sensitive and TH+ sympathetic nerve fibers penetrate the functional layer.

There are no parasympathetic (VAChT+), nitrergic (nNOS+), or somatic (PMP22+) nerve fibers, nor oxytocin+ or RLN2+ fibers.

Innervation of the fetal uterine artery

Innervation of the uterine artery is rich (Supplemental Figure 9). Autonomous innervation of the artery mainly consisted of sympathetic fibers (TH+). Parasympathetic (VAChT+), nitrergic (nNOS+), and sensitive (NPY+, CGRP1+) nerve fibers are also observed.

Three-dimensional reconstruction of fetal uterine innervation

We reconstructed a 3D uterine micro-innervation model focusing on the lower part of the uterus (Figure 3). Nerve fibers penetrate the uterus through the lateral edges of the uterine isthmus. They then follow a centripetal path from the periphery (serosa) to the center (endometrium) with ascending fibers towards the uterine body and descending fibers towards the cervix (Supplemental Figure/video 1).

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DISCUSSION

We present here the first description of the distribution of specific nerves throughout the human uterus accompanied by a comprehensive 3D reconstruction of uterine innervation based on a fetal model. The principal results of this study are that 1) fetal uterine innervation is essentially composed of sympathetic and parasympathetic fibers, and sensitive nerves, 2) all the layers of the uterus are innervated with a centripetal disposition including the endometrium, and 3) nerve fibers penetrate the uterus through the lateral edges of the isthmus.

Mature human fetuses are considered valid and reliable experimental models for pelvic neuroanatomic studies [START_REF] Arango-Toro | Development of the pelvic plexus in human embryos and fetuses and its relationship with the pelvic viscera[END_REF][START_REF] Fritsch | Topography of the pelvic autonomic nerves in human fetuses between 21?29 weeks of gestation[END_REF]. Human fetal tissue has previously been used for pelvic innervation and muscle studies as pelvic innervation is fully developed at 8 weeks of gestation. This was reported in a fetus study by Fritsch [START_REF] Fritsch | Topography of the pelvic autonomic nerves in human fetuses between 21?29 weeks of gestation[END_REF], and later observed by Arango-Toro [START_REF] Arango-Toro | Development of the pelvic plexus in human embryos and fetuses and its relationship with the pelvic viscera[END_REF] in an anatomical study of 15 human embryos and two fetuses ranging in size from 4 to 132 mm crown-rump length: the pelvic plexus was fully developed by Carnegie stage 23. Consequently, pelvic neuroanatomy is stable and comparable with that of the adult after 8 weeks of gestation with no further fundamental changes occurring during fetal growth and post-fetal development. There are several advantages to using fetal tissue. First, the small size of the fetus enables en-bloc pelvis removal and processing, minimizing anatomical data loss, surgical displacement and reconstruction biases compared to adult specimens and surgical dissection. Second, we performed continuous serial sections and were thus able to follow the nerve pathways. Third, nerves are larger in fetuses than in adults proportionally.

Finally, multiple specific muscular and neuronal markers can be used in fresh material.

We used a technique called computer assisted anatomical dissection (CAAD) which was introduced in the 90's by Benoit's team [START_REF] Benoit | Computerassisted prostate reconstruction[END_REF]. This original technique provides an accurate 3D reconstruction of fetal anatomy with identification of the exact organization of the thin nerve elements, their distribution, and their relationship to the vascular, visceral, fascial and muscular structures [START_REF] Alsaid | Computer-assisted anatomic dissection (CAAD): evolution, methodology and application in intra-pelvic innervation study[END_REF]. CAAD is now used by various teams around the world (28-30). In this study, we further improved the basic technique by the addition of co-labelling and immuno-fluorescence. This allowed us to identify several types of nerves as well as uterine smooth muscle in the same image so as to clearly distinguish myometrial from endometrial innervation. Additionally, fluorochromes provided a high discrimination of markings for nerve quantification, which is more objective and compatible with the use of automated nerve quantification software.

11

Few data exist about the micro-innervation of the human uterus as most studies to date have been conducted on animal models [START_REF] Gnanamanickam | Innervation of the rat uterus at estrus: a study in full-thickness, immunoperoxidase-stained whole-mount preparations[END_REF][START_REF] Adham | Autonomic innervation of the rat vagina, cervix, and uterus and its cyclic variation[END_REF][START_REF] Bae | Immunohistochemical study of the distribution of adrenergic and peptidergic innervation in the equine uterus and the cervix[END_REF]. In the present study, we observed that innervation of the myometrium is dense, and that the nerves follow blood vessels or smooth muscle cells. These findings are in agreement with the literature [START_REF] Adham | Autonomic innervation of the rat vagina, cervix, and uterus and its cyclic variation[END_REF]. In 1959, Krantz et al employed the term "nerve plexus" to describe nerves within the human myometrium, based on works dating from the 18 century [START_REF] Krantz | INNERVATION OF THE HUMAN UTERUS*[END_REF]. The presence of endometrial innervation is a matter of debate. In this study, we observed that uterine innervation reached the superficial layer of the endometrium. In most mammalian species, the endometrium has been described as being poorly innervated [START_REF] Gnanamanickam | Innervation of the rat uterus at estrus: a study in full-thickness, immunoperoxidase-stained whole-mount preparations[END_REF][START_REF] Adham | Autonomic innervation of the rat vagina, cervix, and uterus and its cyclic variation[END_REF][START_REF] Bae | Immunohistochemical study of the distribution of adrenergic and peptidergic innervation in the equine uterus and the cervix[END_REF]. In the human uterus, Coupland(34), Krantz, Quinn [START_REF] Quinn | Differences in uterine innervation at hysterectomy[END_REF][START_REF] Krantz | INNERVATION OF THE HUMAN UTERUS*[END_REF] and other authors failed to identify nerves within the basal and superficial endometrium except in pathologic conditions [START_REF] Tokushige | Different types of small nerve fibers in eutopic endometrium and myometrium in women with endometriosis[END_REF][START_REF] Zhang | Innervation of endometrium and myometrium in women with painful adenomyosis and uterine fibroids[END_REF][START_REF] Ellett | Are endometrial nerve fibres unique to endometriosis? A prospective case-control study of endometrial biopsy as a diagnostic test for endometriosis in women with pelvic pain[END_REF][START_REF] Newman | Expression of neuronal markers in the endometrium of women with and those without endometriosis[END_REF].

Few studies have described the specificity of the nerves within the uterus in physiologic conditions. In our study, we observed that uterine innervation was composed exclusively of autonomic nerve fibers. We chose to use specific neurotransmitters to label uterine innervation function as in previous studies: TH+, NPY+ for autonomic sympathetic fibers, and VIP+, NOS+ and VACHT+ for autonomic parasympathetic fibers. Also, as demonstrated by Tokushigue et al, CGRP1+, VIP+ and NPY+ could also be markers for sensitive A delta-and C-type nerve fibers. We found a dense proportion of sympathetic (TH+, NPY+) and parasympathetic (VIP+) fibers in the cervix, and a high proportion of sensitive (CGRP1+) nerves in the uterine body. These results are in concordance with the study by Coupland and Di Tommaso showing that parasympathetic nerve fibers are mainly localized in the cervix( 23) , [START_REF] Coupland | The distribution of cholinergic and other nerve fibres in the human uterus[END_REF]. We also found that the functional layer of the endometrium is already innervated in the female fetus with highly sympathetic (TH+, NPY+) and sensitive (CGRP and NPY+) nerves. Recent studies showing nerves within the endometrium or myometrium in the human uterus in pathologic conditions only identified autonomic sensitive, sympathetic and parasympathetic nerves [START_REF] Tokushige | Different types of small nerve fibers in eutopic endometrium and myometrium in women with endometriosis[END_REF][START_REF] Zhang | Innervation of endometrium and myometrium in women with painful adenomyosis and uterine fibroids[END_REF][START_REF] Ellett | Are endometrial nerve fibres unique to endometriosis? A prospective case-control study of endometrial biopsy as a diagnostic test for endometriosis in women with pelvic pain[END_REF][START_REF] Newman | Expression of neuronal markers in the endometrium of women with and those without endometriosis[END_REF]. This discrepancy with previous studies is related to our specimens of prepubescent uteri. Furthermore, one could speculate that the superficial layer of the endometrium is denervated during puberty secondary to exposure to estrogen [START_REF] Brauer | Estrogen and female reproductive tract innervation: Cellular and molecular mechanisms of autonomic neuroplasticity[END_REF]. We thus suggest that the lack of denervation of the functional endometrium during puberty is a possible nociceptive mechanism.

The function of individual neurotransmitters is poorly understood to date. Sympathetic (TH+, NPY+) nerve fibers may be responsible for endometrial glandular secretion and vasoconstriction. Parasympathetic (VIP+, VACHT+, NOS+) and CGRP1+ are more likely involved in vasodilatation and uterine relaxation.

From a physiologic point of view, dense autonomic uterine innervation is fundamental for uterine function. Unsurprisingly, we observed that uterine innervation was composed exclusively of autonomic nerve fibers with a peri-vascular and peri-muscular organization. This could explain how uterine innervation is involved in contractile activity allowing uterine cyclic peristalsis and uterine contractions during labor. The dense innervation of the cervix that we describe here is coherent with the important role of cervical ripening during labor and menstruation [START_REF] Brauer | Estrogen and female reproductive tract innervation: Cellular and molecular mechanisms of autonomic neuroplasticity[END_REF]. Dysregulation of uterine innervation could affect fertility by disrupting uterine muscle peristalsis and impairing blood flow regulation [START_REF] Newman | Expression of neuronal markers in the endometrium of women with and those without endometriosis[END_REF].

From a clinical point of view, gynecologic pathologic conditions are associated with uterine innervation abnormalities. Endometriotic lesions infiltrate richly innervated anatomic sites, contain abnormal innervation, and develop along the nerves [START_REF] Anaf | Hyperalgesia, nerve infiltration and nerve growth factor expression in deep adenomyotic nodules, peritoneal and ovarian endometriosis[END_REF][START_REF] Tokushige | Nerve fibres in peritoneal endometriosis[END_REF].

Additionally, patients with endometriosis, myomas, or adenomyosis who suffer from pain display innervation of the functional layer of endometrium and a significantly higher innervation of the myometrium than patients without pain(7) ,9,10 . Atwal et al also showed that pelvic pain was associated with abnormal reinnervation and micro-neuromas within the myometrium [START_REF] Atwal | Uterine innervation after hysterectomy for chronic pelvic pain with, and without, endometriosis[END_REF]. Finally, Ellet et al demonstrated that nerve fibers were present in the endometrium of patients with and without endometriosis in a cohort of patients with pain [START_REF] Ellett | Are endometrial nerve fibres unique to endometriosis? A prospective case-control study of endometrial biopsy as a diagnostic test for endometriosis in women with pelvic pain[END_REF]. These results suggest the involvement of endometrial innervation in pain mechanisms rather than in a specific gynecologic disease such as endometriosis. In these pathologic conditions associated with greater innervation density, the role of neurotransmitters can be altered. TH+ and NPY+ enable angiogenic and neurotrophic factors that increase blood vessels and nerve density. VIP+ and CGRP1+ contribute to the inflammatory response.

CGRP1+, VIP+ and NPY+ present in sensitive C and A delta fibers may convey information to the central nervous system about the internal environment and potential noxious stimuli (Table 1).

Despite these results and the use of an original model, our study has some limitations.

First, the use of a fetal model with an immature prepubescent uterus is possibly debatable: hormonal changes during puberty could explain some of the surprising results such as the lack of oxytocin or nNos labeling. According to Bauer et al, there is marked diminution of nerve density at puberty. Additionally, cyclic variations in the levels of sex hormones influence uterine innervation, especially sympathetic nerve fibers [START_REF] Brauer | Estrogen and female reproductive tract innervation: Cellular and molecular mechanisms of autonomic neuroplasticity[END_REF]. Sympathetic nerve fibers express estrogen receptors which suggests that estrogen may regulate their plasticity.

In animal models, increased estrogen levels during the cycle precede the disappearance of uterine sympathetic nerves. During pregnancy, there is also a loss of sympathetic, parasympathetic and afferent nerves secondary to sexual hormone expression (estrogen and progesterone), mechanical strain, hypertrophy of the myometrium and local influence of the placenta [START_REF] Brauer | Estrogen and female reproductive tract innervation: Cellular and molecular mechanisms of autonomic neuroplasticity[END_REF].

There is thus a need for comparative studies of fetal and adult pelvic neuroanatomy. At our center in Rennes, we plan to investigate uteruses from brain-dead donors during multiple organ procurement in the context of a study about uterus transplantation. However, this is the first study to provide a comprehensive model of uterine innervation. Second, the small sample size cannot exclude biases. Nevertheless, the same pattern of uterine innervation was observed in all four fetuses. Moreover, we analyzed the whole uterus avoiding the bias of the variability of innervation of the endometrium. Third, functional relevance is not necessarily associated with clinical relevance. However, the use of neuronal antibodies contributed to the understanding of innervation pathways. Finally, the absence of a somatic uterine innervation may be related to the fact that myelinization is a late event during fetal development. However, using smooth muscle immunostaining, we observed that the whole uterus was smooth and so exclusive autonomic innervation was coherent.

CONCLUSION

We provide here the first comprehensive description of the innervation of the human uterus. This could serve as a basis for future studies on changes in uterine innervation caused by normal physiologic or pathophysiologic conditions.

; 5 )

 5 Incubation of the secondary antibody (OmniMap anti-MsHRP, ref 0526965001-Roche, or OmniMap anti-RbHRP, ref 05269679001-Roche), coupled with an enzyme, the horseradish peroxidase (HRP); 6) Revelation (ChromoMap DAB, ref 05269652001-Roche) and 7) Counter-staining (Hematoxylin II, ref 05277965001-Roche). Immunofluorescence Co-staining was obtained by means of a fluorescent multiplex technique using fluorophoreconjugated tyramide. Three kinds of fluorophores were used: rhodamine (ref 07259883001-Roche), Cyanine 5 (ref 07551215001-Roche) and FAM (ref 07988150001-Roche), and DAPI ((4',6-diamidino-2-phénylindole, ref 0526688001-Roche) which colors the nuclei.

Figure 1 :Figure 2 :Figure 3 :

 123 Figure 1: Illustration of the use of immunofluorescence images to perform nerve quantification with the "HALO" software. Section of the uterine body treated with triple SMA-TH-NPY labeling, with DAPI. Each fluorochrome can be "switched off" or "switched on" depending on what we want to visualize. This illustration shows the centripetal disposition of the innervation of the uterus. A : SMA + TH + NPY + DAPI B : SMA + TH + DAPI C : SMA + NPY + DAPI D : TH + NPY + DAPI E: TH + NPY F : endometrial contouring G : myometrium contouring H : contouring of the serosa I : contouring of the whole uterus

Table 1 :

 1 Summary table describing the neuronal finding and its site, putative physiologic function, and possible pathologic conditions that could lead to patient symptoms (pain, hyperalgesia)

	Neuronal findings	Uterine sites		Putative physiologic function	Pathologic conditions
	PS100:	All layers: very	-	Uterine contractions (labour)	Higher density of nerves in
	All nerves	abundant in the	-	Uterine peristalsis (fertility)	myometrium and endometrium
	Pan neuronal	serosa +++	-	Cervical ripening and opening (labor,	(especially in the functional layer)
				fertility)	Micro-neuroma formation within
		All portions: very			the myometrium, hypertrophy of
		abundant in the			nerve termination
		Cervix +++
	TH :	The most abundant	-	Endometrial glandular secretion	Higher density of nerves
	Autonomic	type	-	regulation of uterine blood flow	enabling angiogenesis.
	sympathetic				Angiogenic and neurotrophic
		Present in all layers			factors increase blood vessels and
					nerve density
		Present in all
		portions: especially in
		the cervix
	NPY :	Very abundant	-	Endometrial glandular secretion	Higher density of nerves in the
	Autonomic		-	Regulation of uterine blood flow	functional endometrium layer
	sympathetic	Present in all layers:	-	Vasoconstrictor (boosts the adrenergic	enabling angiogenic and
		especially		effect)	neurotrophic factors which
		myometrium and			increase blood vessel and nerve
		endometrium			density
					Markers for sensory A delta and
		Present in all			sensory C nerve fibers
		portions: less
		abundant in uterine
		body
	CGRP1 :	Present in all layers:	-	Vasodilatator	Higher density of nerves
	Sensory	especially	-	Smooth muscle cell relaxation	contributing to the inflammatory
	autonomic	myometrium and	-	Inhibition of uterine contractions	response (vasodilation, plasma
		endometrium			extravasation, infiltration by mast
					cells, neutrophils and immune
		Present in all			cells).
		portions: most			Markers for sensory A delta and
		abundant in uterine			sensory C nerve fibers
		body
	VIP :	The least abundant	-	Vasodilatator	Higher density of nerves in the
	Sensory	type	-	Smooth muscle cell relaxation	functional endometrium layer
	Autonomic		-	High uterine blood flow	contributing to the inflammatory
	parasympathetic	Present in all layers:	-	Steroidogenesis	response
		less abundant in the	-	Ovulation,	Markers for sensory A delta and
		endometrium	-	Vaginal lubrification	sensory C nerve fibers
			-	Endometrial glandular secretion
		All portions:
		equivalent abundance
		in the body and the
		cervix
	Nos:	In the present study,	-	Could play a role in maintaining	NO deficiency contributes to the
	Autonomic	only seen		uterine quiescence during pregnancy	hypertensive disorder of
	Parasympathetic	surrounding uterine	-	Powerful inhibitor of platelet	pregnancy: preeclampsia
	Pro-erectile nerve	artery	-	Contributes to maternal systemic
	bundles			vasodilation during pregnancy,
				regulates uterine and fetoplacental
				blood flow
			-	Degree of menstrual bleeding
			-	Might participate in the initiation and
				control of menstrual bleeding
	Vacht:	In the present study,	-	Associated with myometrial and	Activation of sensory C nerve
	Autonomic	only seen		vascular smooth muscle	fibers through
	parasympathetic	surrounding uterine	-	Induces contraction of the	nicotinic receptor neurons.
		artery		myometrium
		Myometrium
		Cervix

Acknowledgements:

The authors want to express their gratitude to the parents who consented to donate a fetal body to science after late miscarriage.

The authors want also to acknowledge Jean-Eric Berton and Julien Landreau for the technical support.

Supplemental Materials

Supplemental Table 1: The red ellipses indicate areas of markings characteristic of a "background noise" in immunofluorescence.

The anti-S100, TH, NPY, CGRP1 and VIP immunostaining is positive, and the antioxytocin marking is presented as an example of negative marking with "background noise". The other labels not shown were all negative.

Supplemental Figure 4: Graph representing the nerve density in each portion and layer of the uterus (in % of the studied surface) calculated with the "HALO" software.

The density of nerve fibers is the highest in the supra-vaginal portion of the cervix, and higher in the serosa in periphery. Nerve fibers are rare but present in the endometrium.

Supplemental