#,⸸ Li-Bo Huang

Fumihiko Mamiya

Marc Baaden

Eiji Yashima

Mihail Barboiu

Self-Assembling Peptide-Appended Metallomacrocycle Pores for Selective Water Translocation

Keywords: artificial water channels, porins, self-assembly, water clusters, macrocycles

Artificial water channels-AWCs selectively transport water, excluding all ions. Unimolecular channels been synthesized via complex synthetic steps. Ideally, simpler compounds requesting less synthetic steps should efficiently lead to selective channels by self-assembly. Herein, we report a self-assembled peptide-bound Ni 2+ -metallomacrocycle, 1, in which rim-peptide-bound units are connected to a central macrocycle obtained via condensation in the presence of Ni 2+ ions. Compound 1 achieves a single-channel permeability up to 10 7 -10 8 water/s/channel and insignificant ion transport, which is one order of magnitude lower as for Aquaporins. Molecular simulations probe that sponge-like aggregates can form to generate transient cluster water pathways through the bilayer. Altogether, the adaptive metallo-supramolecular self-assembly is an efficient and simple way to construct selective channel superstructures.

INTRODUCTION

The selective transport of metabolites across bilayer membranes in living cells is facilitated by simple molecular carriers or by complex protein channels. [START_REF] Saier | Families of Proteins Forming Transmembrane Channels[END_REF][START_REF] King | From Structure to Disease: The Evolving Tale of Aquaporin Biology[END_REF][START_REF] Cukierman | Proton Mobilities in Water and in Different Stereoisomers of Covalently Linked Gramicidin A Channels[END_REF] Biomimetic artificial channels have been designed to understand the translocation mechanisms of ions and water molecules through the membranes and used as active components of sensing, separation and delivery devices. [START_REF] Sisson | Synthetic Ion Channels and Pores (2004-2005[END_REF] Single molecule spanning the bilayer membranes, have been used in an early stage by the researchers pioneering the field, to construct synthetic channels. [START_REF] Lehn | Physical Chemistry of Transmembrane Ion Motions[END_REF][START_REF] Gokel | Synthetic Organic Chemical Models for Transmembrane Channels[END_REF][START_REF] Otis | How Far Can a Sodium Ion Travel within a Lipid Bilayer?[END_REF][START_REF] Jullien | App M l l Channels Synthesis of a Macrocycle-Based Molecular Bundle[END_REF] They are positioned as a macrocyclic scaffolding relay in the middle of the membrane, whereas functional groups attached to this central relay form bundles, closing a unimolecular pillared channel. Significant progress has recently been obtained in this field, when Hou et al. realized that pillar[x]arenes x = 4,5 -PA can be used as a scaffolding relay, [START_REF] Hu | Single-Molecular Artificial Transmembrane Water Channels[END_REF][START_REF] Chen | Chiral Selective Transmembrane Transport of Amino Acids through Artificial Channels[END_REF][START_REF] Si | Voltage-Driven Reversible Insertion into and Leaving from a Lipid Bilayer: Tuning Transmembrane Transport of Artificial Channels[END_REF] whereas functional groups attached to aromatic rings form unimolecular pillared channels, replacing natural aquaporins in cells. [START_REF] Yan | Artificial Aquaporin That Restores Wound Healing of Impaired Cells[END_REF] We recently described that dimerization of rimdifferentiated PAs, produces expanded channels with osmotic water permeability close to natural aquaporins. [START_REF] Strilets | Biomimetic Approach for Highly Selective Artificial Water Channels Based on Tubular Pillar[5]arene Dimers[END_REF][START_REF] Huang | Artificial Water Channels: Towards Biomimetic Membranes for Desalination[END_REF] Another attractive strategy for the construction of biomimetic channels is based on the self-assembly of several building blocks towards the creation of supramolecular channels. [START_REF] Zheng | Self-Assembled Artificial Ion-Channels toward Natural Selection of Functions[END_REF] Most of the reported channels are composed of organic molecules self-assembled via H-bonds. [START_REF] Barboiu | An Artificial Primitive Mimic of The Gramicidin-A Channel[END_REF]17 Metallosupramolecular architectures, presenting ionic conductance states in bilayer membranes have been reported during the last decade. [START_REF] Zheng | Self-Assembled Artificial Ion-Channels toward Natural Selection of Functions[END_REF] Fyles et al. [START_REF] Fyles | Predicting Speciation in The Multi-Component Equilibrium Self-Assembly of a Metallosupramolecular Complex[END_REF] was pioneering the field and incipiently demonstrated that large supramolecular channels with internal pore diameters of 10 nm can be assembled from organic building blocks and metal cations. They started to construct such scaffolds based on a lipop l F j ' pl x [START_REF] Fujita | Preparation of A Macrocyclic Polynuclear Complex, [(en)Pd(4,4'-bpy)]4(NO3)8 (en = Ethylene Diamine, bpy = Bipyridine), Which Recognizes an Organic Molecule in Aqueous Media[END_REF] but it was shown that the lipophilic pore is more complex and is composed of several Pd 2+ centers in the bilayer membrane. This was the starting point to explore other functional channels based on metal-organic complex polyhedra. [START_REF] Jung | Synthetic Ion Channel Based on Metal-Organic Polyhedra[END_REF][START_REF] Kawano | Metal-Organic Cuboctahedra for Synthetic Ion Channels with Multiple Conductance States[END_REF][START_REF] Mahon | Biomimetic Approach for Ion Channels Based on Surfactant Encapsulated Spherical Porous Metal-Oxide Capsules[END_REF][START_REF] Kulikov | A Synthetic Ion Channel Derived from a Metallogallarene Capsule That Functions in Phospholipid Bilayers[END_REF] This simple and effective strategy may conveniently demonstrate the potential of using simple molecules and metal ions to easily generate biomimetic nanopores with ion channel activity in lipid bilayers, avoiding complex synthetic steps required by the design of single-molecular channel.

EXPERIMENTAL SECTION

Transmembrane transport experiments Sample preparation. We set the concentration gradients (15 20 25 3 μM esponding to the molar channel to lipid ratio (mCLR) = 0.034, 0.046, 0.058 and 0.070 mol%. In the cation transport HPTS (pyranine, 8-hydroxypyrene-1,3,6trisulfonic acid trisodium salt) assays, we added complex 1 by premixing method when preparing LUV, and the final concentrations of complex 1 in LUV suspension were 0.14, 0.17 0.22 0.25 d 0.28 μM corresponding to mCLR = 0.025, 0.030, 0.040, 0.045 and 0.050 mol%. It is noteworthy that the composition of LUV used in water transport assays and cation transport assays was different. Water transport by using D(+)-sucrose as osmolyte. LUV were prepared using the film rehydration method. A phosphatidylcholine, phosphatidylserine and cholesterol PC/PS/Chl mixture with a molar ratio of 4/1/5 was dissolved in chloroform/methanol mixture (CHCl3/MeOH, v/v: 1/1). The solution was dried on a rotary evaporator and subsequently in a vacuum desiccator to remove residual solvent. After rehydration with 1 mL buffer containing 200 mM sucrose and 10 mM PBS (pH = 6.4), the suspension was extruded through 100 nm track-etched filters for 21 times (Whatman, UK) to obtain monodisperse unilamellar vesicles, the size of which was characterized by dynamic light scattering (Zetasizer Nano, Malvern Instruments Ltd., UK). The aliquots of complex 1 dissolved in DMSO have been added to the LUV. The water permeability tests were conducted on a stopped-flow instrument (SFM3000 + MOS450, Bio-Logic SAS, Claix, France). Exposure of vesicles to hypertonic osmolyte (300 mM sucrose) in the same buffer resulted in the shrinkage of the vesicles due to an outwardly directed osmotic gradient. The changes of light scattering were recorded at a wavelength of 345 nm. The abrupt change of the vesicle size led to variation in the light scattering at 90° according to the Rayleigh-Gans theory applied to this system and could be fitted in the form of the sum of double-exponential functions. The osmotic permeability (Pf) was calculated as following:

where k is the smaller exponential coefficient (k1) obtained by fitting the light scattering traces into double-exponential functions, because the larger exponential coefficient (k2) showed no apparent functional relationship with increasing permeability in these assays; S and V0 are the initial surface area and volume of the vesicles, respectively; Vw l l f w d Δosm is the osmolarity difference.

In an average exp 100 μL k l p d l g 0.524 g f l p d w d l d w 1880 μL f 200 mM sucrose or 115 mM NaCl in 10 mM PBS buffer solution. The complex 1 w j d 20 μL l DMSO. The solution mix was maintained at 20 ℃ for 30 minutes before exposure to the 400 mM sucrose solution.

Experiments have been conducted at different concentrations of the injected complex 1, in which the mCLR of complex 1 in LUV suspension were 0.034, 0.046, 0.058 and 0.070 mol%. Average permeability was calculated by using the permeability values obtained from at least three different batches of vesicles. Single-channel permeability calculations. One complex is needed to form a channel within a transmembrane l g f 5 . k g pl 0.20 μM as an example, the molar channel to lipid ratio is 0.046 mol%. The average radius of the liposome was 72.5 nm determined by DLS experiments. Hence, the sum of outer and inner surf w 4π × 2 + 4π × ( -5) 2 = 123,308 nm 2 . The average cross-sectional area of a lipid in average was 0.287 nm (PC/PS/Chl lipids with a molar ratio of 4/1/5: the cross-sectional areas of PC and PS were ~0.35 nm 2 and that of cholesterol was 0.223 nm 2 ), and that of the channel was estimated as 2.80 nm 2 . So, the insertion number of the channel was ~98 per vesicle. If the overall net permeabily by l l p w (3.05 ± 0.47) μ / single-channel permeability was (1.91 ± 0.29) × 10 -15 cm 3 /s and (6.38 ± 0.98) × 10 7 water molecules/s. According to the same algorithm, the single-channel permeabilities for the channels at different mCLR were shown in Table S1 andS2. The single-channel permeability values covered the range of 1.8 × 10 7 -6.4 × 10 7 H2O/s/channel, which are within the same order of magnitude as that of Aquaporins (~10 8 -10 9 water molecules/s/channel). Water transport by using NaCl as osmolyte. The preparation of liposomes and the stopped-flow assays were the same as the previous method for water transport in sucrose solution. To implement the same osmotic pressure (106 mOsmol/kg), the concentration of NaCl osmolyte was different from D(+)-sucrose. Internal: 115 mM NaCl and 10 mM PBS, pH = 6.4; External: 172.5 mM NaCl and 10 mM PBS, pH = 6.4 (after mixture). Cation transport HPTS assays.

LUV preparation. The complex 1 and egg yolk L-phosphatidylcholine (EYPC chloroform solution, 800 µL, 26 mmol) were dissolved in a CHCl3/MeOH mixture, the solution was evaporated under reduced pressure and the resulting thin film was dried in a vacuum desiccator for 2 h. The lipid film was hydrated in 400 µL phosphate buffer (10 mM sodium phosphate, pH = 6.4, 100 mM NaCl) containing 10 µM HPTS for 40 min. During hydration, the suspension was submitted to at least 7 freeze-thaw cycles (bathed in liquid nitrogen and water at room temperature, respectively). The large multilamellar liposome suspension (1 mL) was submitted to high-pressure extrusion at room temperature (21 extrusions through a 100 nm polycarbonate membrane afforded a suspension of LUVs with an average diameter of ~100 nm). The LUV suspension was separated from extravesicular dye by size exclusion chromatography (SEC) (stationary phase: Sephadex G-50, mobile phase: phosphate buffer) and diluted to 2.8 mL with the same PBS buffer to give a stock solution with a lipid concentration of 9.3 mM (assuming 100% of lipid was incorporated into liposomes). Cation transport. 100 µL HPTS-loaded vesicles (stock solution) was suspended in 1.86 mL sodium phosphate, pH = 6.4 with 100 mM MCl (M = Na + , K + , Rb + or Cs + ) and placed into a quartz fluorimetric cuvette. The emission of HPTS at 510 nm was monitored with excitation wavelengths at 403 and 460 nm simultaneously, using a Perkin Elmer LS-55 fluorescence spectrometer. During the experiment, 29 µL of 0.5 M aqueous NaOH was injected at t = 20 s, resulting in the pH value increasing to around 7 in the extravesicular media. For the reference proton transport experiment, 20 µL of 50 µM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) solution in DMSO was added at t = 10 s. Maximal possible changes in dye emission were obtained at t = 320 s by lysis of the liposomes with detergent (40 µL of 5 % aqueous Triton X100). We expressed normalized intensity (the value of I460/I405) as a function of time from t = 0 s to 320 s. To calculate EC50 and Hill coefficient n, we used the fractional activity Y. Y was calculated for each curve using the normalised value of I460/I403 (just before lysis of the vesicles), from 0 (ratio for the blank) to 1 (highest ratio obtained, i.e. a plateau). We expressed Y as a function of time, and we performed fittings using a 2-parameter equation, which is, Hill equation:

The Hill coefficient is an indication the cooperativity of the channel forming elements. There is two ways to find Hill coefficient n and EC50: either fitting of the experimental curve using the Hill function (as we did), or using the expression:

where n is the slope of the curve and n*log (EC50) is the log(C) = 0 intercept value.

Molecular dynamics simulations of compound 1 channels in a fully hydrated lipid bilayer environment.

To prepare the starting point used for the simulations, the macrocycle had to be subdivided into smaller "residues", 3 identical ones for the cycle itself, 6 for the core of the branched R chains and 6 for the terminal OTG groups as illustrated in Figure 3a. We also set up a 7-mer aggregate in the membrane as simulation C1-NI2-x7, using our own virtual reality tools to assemble a tight initial complex from a pre-equilibrated monomer along the protocol described,28 which was run for 105 ns. During the equilibration of the simulations we manually wetted the channel interior to stabilize the hollow structure, and attempted to fix atom positions of the macrocycle and the included waters in the beginning of the equilibration period.

The C1-NI2 simulation was set up automatically by aligning the macrocycle axis with the membrane normal, which was then oriented along the Y-axis, normally with respect to the plane of the membrane and the XZ-plane. Having placed an equilibrated membrane structure (consisting of phosphatidyl-choline molecules) at this location named 'MemCenterY', the system was enclosed in a simulation cell of size 65*63*65 Å, the macrocycle was temporarily scaled by 0.9 along the XZ-axes, then strongly clashing membrane lipids were deleted (lipids with an atom closer than 0.75 Å to a macrocycle atom). The temporary macrocycle scaling, which was needed to avoid the deletion of too many lipids around the protein, was then slowly removed during a short simulation at 298K in vacuo: the macrocycle (with all atoms kept fixed) was scaled by 1.02 along the XZ-axes every 200 femtoseconds, while the membrane was allowed to move, but restrained to ideal geometry (by pulling lipid residues with an atom further than 21.5 Å away from MemCenterY back into the membrane, and by pushing phosphorus atoms closer than 14 Å to MemCenterY back outwards). The force field was AMBER14, with Lipid14/GAFF/AM1BCC parameters for non-standard residues. As soon as the macrocycle had reached its original size again, the simulation cell was filled with water, 0.9% NaCl and counter ions The main simulation was then run with PME and 8.0 Å cutoff for nonbonded real space forces, a 4 fs time-step, constrained hydrogen atoms, and at constant pressure and temperature (NPT ensemble), as described in detail previously. During the initial 250 picoseconds, the membrane was restrained to avoid distortions while the simulation cell adapted to the pressure exerted by the membrane (see above, additionally water molecules that got closer than 14 Å to MemCenterY were pushed outside). The source code of this simulation protocol and visualizations of the individual steps can be found at www.yasara.org/membranemd. C1-NI2-x7 was set up in a similar way, with a larger initial cell size.

RESULTS AND DISCUSSION

Design strategy. Herein we concentrate our efforts on investigation of water/ion transport properties through lipid bilayer membranes containing self-assembled discrete peptide-bound Ni 2+ -salphen-based metallomacrocycle, 1 (Figure 1), in which rim-peptidebound phenylene units are connected to a central metallomacrocycle. Tubular channel superstructures are obtained through self-assembly via multiple imine/coordination reactions of the peptide-bound disalicylaldehyde with ortho-phenylenediamine in the presence of Ni 2+ acetate. The synthesis previously reported by Yashima et al. is quantitatively leading to the trimeric macrocycle in high yields (92-97%). [START_REF] Mamiya | Remote Control of the Planar Chirality in Peptide-Bound Metallomacrocycles and Dynamic-to-Static Planar Chirality Control Triggered by Solvent-Induced 3(10)-to-α-Helix Transitions[END_REF] The unprecedented dynamic planar chirality observed with compound 1 is crucial for maintaining the pR/pS interconversion in this system while it might be strongly suppressed when the compounds is embedded in a bilayer membrane environment.

The hydrophobic nature of the peptide arms may provide not only an important solubility of these compounds in the membrane, but more important a rational design strategy for developing macrocycle-based ion channels that make use of 3 [START_REF] Chen | Chiral Selective Transmembrane Transport of Amino Acids through Artificial Channels[END_REF] -to-a-helix transitions to regulate the opening of the pores and implicitly the selectivity of the transport. [START_REF] Mamiya | Remote Control of the Planar Chirality in Peptide-Bound Metallomacrocycles and Dynamic-to-Static Planar Chirality Control Triggered by Solvent-Induced 3(10)-to-α-Helix Transitions[END_REF] Other hydrophobic peptides scaffolded around synthetic pillarene [START_REF] Hu | Single-Molecular Artificial Transmembrane Water Channels[END_REF][START_REF] Chen | Chiral Selective Transmembrane Transport of Amino Acids through Artificial Channels[END_REF][START_REF] Si | Voltage-Driven Reversible Insertion into and Leaving from a Lipid Bilayer: Tuning Transmembrane Transport of Artificial Channels[END_REF][START_REF] Yan | Artificial Aquaporin That Restores Wound Healing of Impaired Cells[END_REF] or cyclodextrins [START_REF] Hjørringgaard | Cyclodextrin-Scaffolded Alamethicin with Remarkably Efficient Membrane Permeabilizing Properties and Membrane Current Conductance[END_REF] have been developed and inserted into bilayers.

Interestingly, the analysis of an energy minimized structure of 1 shows the formation of a dynamic bundle-type unimolecular channel architecture. This scaffold might allow the translocation of partly dehydrated cations or water molecules along well-fitting membrane inserted channels of 4.0 nm theoretical length (Figure 1b), formed via self-assembled double-faced lateral peptide arms anchored to a large central pore of 1.2 nm diameter (Figure 1c). We anticipated that closely spaced peptide appended metallomacrocycle 1 would allow interactional transloca-tion, restriction and control of the transport activity within a central selectivity filter, as for natural proteins.

Water-Transport Experiments. The peptide-bound Ni 2+ -salphen-based macrocycle channels, 1, were reconstituted into lipid vesicles (100 nm in diameter) of phosphatidylcholine/phosphatidylserine/cholesterol (PC/PS/ Chl, 4/1/5, molar ratio) containing 200 mM D-(+)sucrose or 115 mM NaCl in 10 mM PBS, buffer solution (pH = 6.4). The complex 1 was added from their DMSO solutions to pre-formed vesicles. Then, the vesicles were exposed to outwardly hypertonic conditions driven by 10 mM PBS buffer solution (pH = 6.4) of 300 mM D-(+)sucrose or 172. Unfortunately the experimental water transport rates are not very high, however interesting improvements for initial rates were observed when the water channels are added in the membrane. If such water transport rates are very close, then a first order fitting method will give you an accurate value of an improved water rate. The k value for water transport can be then obtained by subtracting the control rate. The water transport rates are strongly de-pendent on the molar channel to lipid ratio (mCLR) of 1: 0.034, 0.046, 0.058, 0.070 mol%, that increase the net water permeability relative to the background lipid permeability in the bilayer membrane (Figures 2a). It can be noticed that with 100 mM D-(+)-sucrose osmolarity (osm = 106 mOsmol / kg) the net channel permeabilities remain l l f g d ~2 μ / f ll ations (Figure 2a). The single channel permeability values covered the range of 1.8 × 10 7 -6.4 × 10 7 H2O/s/channel and they are decreasing with increasing concentration. The net channel permeabilities are improved and show a tend y f g p ~7.62 μ / w 57.5 M l osmolarity (osm = 106 mOsmol/kg) (Figure 2b). The increasing permeabilities values in the presence of metallomacrocycle, 1 are interesting, however they are not out competing the natural water translocating counterparts.

Cation and Proton-Transport Experiments Cation (M + = Na + , K + , Rb + and Cs + ) transport activities across the bilayer membranes incorporating channels 1, reconstituted into phosphatidylcholine (PC) lipid vesicles (100 nm), were assessed using standard HPTS fluorescence assays (Figure 2c,d, S1-S5). [START_REF] Matile | Analytical Methods in Supramolecular Chemistry[END_REF][START_REF] Li | Structure-Driven Selection of Adaptive Transmembrane Na + Carriers or K + Channels[END_REF] Indeed, when tested with MCl on external buffer, the channel 1 does not present a doseresponse-type activity, showing very low (< 10%) or near to zero activity behaviors (Figure 2c), reminiscent with negligeable M + /H + antiport conductance states of the channels 1 in the membrane. This confirms our initial assumption a restricted transport of the hydrated cations may occur through the hydrophobic sterically restricted pore formed by the peptide arms. In order to verify if the H + transport is the rate-limiting step for M + /H + antiport activity, we conducted HPTS assays with compounds 1 in the presence of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone, FCCP as a H + carrier (Figure 2d, S1-S7). [START_REF] Li | Structure-Driven Selection of Adaptive Transmembrane Na + Carriers or K + Channels[END_REF] These experiments have shown a difference with the previous results suggesting increased cation transport activities for all MCl salts in the presence of 0.14 to 0.28 µM of 1. Among them, the lowest value of EC50(K + ) = 17.4 µM is observed for K + (Table S3). However the transport selectivity for K + is very low when compared with other cations : EC50(Na + ) = 20.9 µM, EC50(Rb + ) = 19.5 µM, EC50(Cs + ) = 20.9 µM. Hill coefficients are for all the cations from 1.5 to 2 and are indicative of a high degree of cooperativity for cation transport. On this basis, the resulting rather weak transport selectivity, indicates that the transport of the hydrated anions is slightly governed by the solvation free energy of the cations and most probably related to a competitive Cl -/H + symport. Molecular dynamic simulations. Three molecular dynamics simulations of compound 1 were performed in a fully hydrated membrane environment to probe its capacity to form artificial water channels and transport water across membranes. First, we examined how a single instance of compound 1 behaves in a membrane environment of pure POPC. For the simulation, the axis of the macrocycle was aligned with the membrane normal, which was then oriented along the Y-axis, perpendicular to the plane of the membrane coincident with the XZ-plane (Figure 3b left). The simulation shows the rapid reorientation of the macrocycle in the membrane (Figure 3b right).

In isolation, it is unstable when starting with its central ring horizontally aligned in the membrane. This leads to a general skew of the whole compound within the membrane due to ring tilting, which restricts the internal channel space. This observation was confirmed by two independent 20 ns simulations, which indicated variable flexi-bility of the compound. The central macrocycle remained fairly fixed in the tilted position, whereas the peptide-PEG sidechains exhibited considerable flexibility, with some PEG chains pending in water (Figure 3c). The experimental data showed that transport performances are concentration dependent, so the aggregation on the metallomacrocycle 1 might play a role using virtual reality, [START_REF] Baaden | Virtual Reality@Intelligent Hardware[END_REF] we then built a larger assembly of 6 instances of 1 arranged in a hexagon around a central instance. This simulation was run for more than 100 ns, resulting in an irregular membrane-inserted aggregate producing spongelike water passages (Figure 4).

Overall, all simulation results indicate that membranespanning hydration can be maintained and stabilized by the inserted metallomacrocycle 1, although the conformation of the molecule is distorted by membrane pressure, aggregation, and out-of-plane orientation of the cycle (Figure S8). Figure 3d shows the hydration of the compound throughout the simulation. The water structure stabilizes with time, along the entire length of the membrane despite the unexpected asymmetric position and orientation of the compound within the membrane (SI Movie). We observed a few water passages through the channel core and many permeation events through the sponge-like water network ramifications at the interfaces between the instances of compound 1 and the lipid environment.

The orientation of the molecules reflects a porous structure and the initial transient appearance of water clusters inside the channel is in the region of the peptide lateral arms followed the water cluster stabilization in the central metallomacrocycle 1 pore interacting via Ni 2+ salphen centers. The channel preference to form interconnected channel---H2O---H2O networks is the major factor determining their water / ion permselectivity. Contrary to carrier ions diffusion, proton translocation via directional H-bonds of water clusters is a structural diffusion and is strongly depending on the water cluster stabilization, which seems to be still very dynamic as observed in experimental proton translocation studies. [START_REF] Decoursey | Voltage-Gated Proton Channels and Other Proton Transfer Pathways[END_REF] The metallomacrocyclic 1 channel architecture is a very intriguing system. It is very tempting to equate that permeable water cluster sponge-like constructs are stabilized in the bilayer membrane. The assumption is that dynamic H-bonding with lateral peptide arms and hydrophobic interactions would initiate the water cluster formation, providing further premises for the stabilization of water cluster translocation pathways through the central metallo-macrocycle via Ni 2+-salphen centers in the center of the membrane. The water cluster stabilization confers to these channels self-protective behaviors against ions, as the cation/anion insertion will be energetically unfavorable, impeding the water cluster formation.

CONCLUSIONS

In conclusion, we have demonstrated here that selfassembled metallomacrocyclic-channels 1 can be obtained from salphen-peptide decorated ligands and Ni 2+ metal ions present enhanced selective water permeability with almost total ion-rejection across a lipid bilayer. Slight tilted orientation of the macrocycle in the membrane and the very dynamic disposition of peptide lateral arms surrounding the macrocyclic cavity of the channels make them highly adaptive of transporting water at a rate one order of magnitude lower as for the natural AQP. These values are practically within the same order of magnitude ~10 7 water molecules/s/ channel as those observed for other pillararenes channels reported by Hou et al. [START_REF] Yan | Artificial Aquaporin That Restores Wound Healing of Impaired Cells[END_REF][START_REF] Xiao | Artificial Water Channel that Couples with Cell Protrusion Formation[END_REF] or by us [START_REF] Strilets | Biomimetic Approach for Highly Selective Artificial Water Channels Based on Tubular Pillar[5]arene Dimers[END_REF] or hydroxy channels. [START_REF] Huang | Hydroxy Channels-Adaptive Pathways for Selective Water Cluster Permeation[END_REF][START_REF] Mondal | Selective and rapid water transportation across a selfassembled peptide-diol channel via the formation of a dual water array[END_REF] More importantly, salt rejection and the extremely low proton permeability through hydrophobically sterically restricted channels, offer the closest functional mimic of the natural AQP through a synthetic scaffold. These channels can be easily synthesized via simple self-assembly processes from simple components and are the first ones of this class of metallosupramolecular channels showing selective water permeation. Within this context, the metallomacrocyclic channels reported here may be considered as a novel important milestone on the way to the systematic discovery of synthetically accessible artificial water channels. They open up new directions and perspectives in AWCs research towards the construction of biomimetic membranes for desalination. [START_REF] Huang | Artificial Water Channels: Towards Biomimetic Membranes for Desalination[END_REF]

ASSOCIATED CONTENT

Supporting Information

This material is available free of charge at the ACS Publications website.

Details about kinetic data of water permeabilities using D(+) sucrose and NaCl as Osmolytes. M + = Na + , K + , Rb + and Cs+ ion transport activities expressed as fluorescence intensities at different concentration of 1. Hill analysis results of complex 1 with FCCP. Initial and final snapshot of the molecular simulation after 20 ns.

Table of Content

Figure 1 .

 1 Figure 1. (a) Chemical formula and (b) lateral and (c) top views of the energy minimized structures of pep-tide-bound Ni2+-salphen-based metallomacrocycle, 1.

  30 μM) f pl x 1 l DM O f water transport assays. When injected into large unilamellar vesicles (LUV) suspension, the final concentraf pl x 1 b 0.15 0.2 0.25 d 0.

  5 mM NaCl osmolytes, to evidence the shrinkage of the liposomes that increased the lightscattering signal (Figure 2a,b).

Figure 2 .

 2 Figure 2. Water net permeability and single-channel permeability values determined at different concentrations of channels in the liposomes by using stopped-flow light scattering experiments and (a) D-(+)-sucrose or (b) NaCl as osmolytes. Comparison of M + = Na + , K + , Rb + and Cs + transport activities expressed as normalized fluorescence intensity of 0.14 µM (0.025 mol%) channels in the extravesicular media containing 100 mM MCl, (c) in the absence or (d) with the presence of FCCP as H + carrier. Maximal possible dye emission is obtained at t = 320 s by lysis of the liposomes with detergent Triton X100.

Figure 3 .

 3 Figure 3. (a) Schematic initial side view of the metallomacrocycle 1 with the components highlighted by different colors, embedded in a fully hydrated POPC (1-Palmitoyl-2-oleoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine) membrane. Parts of

Figure 4 .

 4 Figure 4. (a) Simulation of a 7-mer of compound 1 (in blue), starting from the arrangement shown at t=0 ns (looking from above the membrane down the membrane normal) toward a cumulative view from 85 to 105 ns to illustrate flexibility. (b) Different views of the water network (water in white and red, surface and hydrogen bonds in pink). The POPC membrane is shown by the connectivity of the phosphorus atoms of the individual leaflets (in white). Parts of water, ions and lipids have been omitted for clarity.

ACKNOWLEDGMENT

The authors wish to acknowledge the Agence Nationale de la Recherche for financial support (ANR-22-CE06-0024-02, BIOWATER)

AUTHOR INFORMATION Corresponding Authors

Mihail Barboiu


Iuliana-Marilena Andrei

Dr Dmytro Strilets

Dr Shixin Fa

Marc Baaden

Tomoki Ogoshi
email: ogoshi@sbchem.kyoto-u.ac.jp

Mihail Barboiu
email: mihail-dumitru.barboiu@umontpellier.fr

I.-M Andrei

Prof. T Ogoshi

Combinatorial screening of water/proton permeation of selfassembled Pillar[5]arene artificial water channel libraries

Binary combinations of pillararenes libraries have been used to construct selective artificial water channels presenting increased water transport activities across lipid bilayers than former components A-D.

Abstract: Artificial water channels-AWCs that selectively transport water rejecting ions through bilayer membranes have potential to act as synthetic Aquaporins (AQPs). AWCs can have a similar osmotic permeability, better stability, with simpler manufacture on a largerscale and have higher functional density and surface permeability when inserted into the membrane. Here, we report the screening of combinatorial libraries of symmetrical and unsymmetrical rim functionalized PAs A-D, that are able to transport ~10 7 -10 8 water molecules/s/channel, which is within 1 order of magnitude of AQPs' and show total ion and proton rejection. Among four channels, C and D are 3-4 times more water permeable than A, B when inserted in bilayer membranes. The binary combinations of A-D with different molar ratios could be expressed as an independent (linear ABA), a recessive (inhibition AB, AC, DB, ACA) or a dominant (amplification, DBD) behavior of the water net permeation events.

Cells are the building blocks of any living organism, separating the intracellular space from the extracellular environment. From a functional point of view, the biological membranes have one of the most important functions of regulating the metabolites' transport for the cell membrane through natural protein channels. [1] Water translocation across membranes is an important physiological process that can be selectively produced at accelerated rates via protein water-selective channels, such as Aquaporins-AQPs. [2,3] Over time, the investigation of the water transport mechanism through natural AQP channels inspired the researchers into developing biomimetic Artificial Water channels-AWCs, involved in transmembrane water transport with total ion rejection. [4][5][6][7][8] A particular interest in the construction of self-assembled supramolecular channels for selective transport of water across the bilayer membranes are the easily-accessible Pillar [5]arenes (PAs) synthesized for the first time by one of us. [9] PAs have a highly symmetrical pillar-shaped structure, tunable size of cavity, fine-tunable rims with easy functionalization. Hou et al. proposed them as suitable platforms to construct artificial water channels. The formation of columnar structures could be induced by the intramolecular hydrogen bonding between the side-chains on pillar [5]arenes. [5,[10][11][12][13] Since their discovery, the water transport properties of peptide-PAs, as a bioinspired approach for artificial water channels, have been intensively studied for their high water permeability similar to that of AQPs. [START_REF] Shen | Proc. Natl. Acad. Sci[END_REF][START_REF] Li | [END_REF][16] Another step into the design approaches for their construction are the selfassembly of rim-differentiated Pillar [5]arenes as expanded length-controlled tubular platforms for host-guest recognition materials or for the construction of directional channels within lipid bilayer or polymeric membranes. [17][18][19][20][21][22][23] The natural AQPs are known to transport water at high rates (~10 8 -10 9 water water/s/channel) in a selective manner with a narrowest constriction active pore filter of 2.8 Å, that selectively accommodate and transport water wires. [24][25][26] The tubular pillar [5]arenes have often an internal cavity ~5 Å, which acts as highly water permeating, but non-selective platform for single water-wire translocation over ions. [START_REF] Shen | Proc. Natl. Acad. Sci[END_REF]27,28] Recently, we found that highly selective artificial water-channels may be obtained by self-assembling pillar [5]arene dimers, presenting fast transport of water and offering size restriction selectivity, unprecedently observed with the PA channels before. [29] Within these PAs, classical relays of 5 Å diameter are superposed with a narrowest twisted hydrophobic pore of 2.8 Å diameter. [30] This affords a size restricted control of water translocation along tubular channels at narrowest selectivity filter level, offering size restriction, similar to natural protein channels. Hou et al showed that positive charge groups at the entrances indicates that the positive charges enhance water transport. [31] Pursuing these endeavors to conceive original and efficient water channels, herein we concentrate our efforts on investigation of water/ion transport abilities through bilayer lipid membranes of combinatorial libraries of dynamic self-assembled mixtures of rim-differentiated peralkyl-carboxylate-pillar [5]arene, A and peralkene-carboxylate-pillar [5]arene, D and symmetrical rim-functionalized peralkylamino-substituted pillar- [5]arene, B and percarboxylate-pillar- [5]arene, C (Figure 1a). The water transport properties are dependent on the nature of the functional groups grafted onto the PA platform as certain dimeric and trimeric combinations at different molar ratios (1:1 and 2:1) (Figure 1b), can be obtained from mixtures of mono-(A, D) and bis functional (C) negatively charged carboxylates or bisfunctional positively charged ammonium (B) pillar [5]arenes that increase the overall water permeability relative to the single pillar [5]arenes components. The self-assembly of lengthcontrolled discrete dimeric or trimeric tubular superstructures by homo-/hetero-/co-assembly via hydrogen bonds and salt bridges of rim-differentiated A and peralkylamino B pillar [5]arenes have been previously demonstrated by Ogoshi et al. [29] by using NMR spectroscopy. Molecular simulations of the single crystal structure of dimers A2 embedded in bilayer membranes showed that the dimer is stable in the membrane, leading either to tilted or shifted side-by-side aggregated membrane-embedded structures, none of them featuring any water permeation. [30] So we tested whether larger assemblies of honeycomb arrangement where a central dimer is surrounded by six other dimers, provided increased stabilization within the membrane environment. [30] The honeycomb aggregate initiates water permeation events through the dimeric pore assembly with a continuous water accumulation around carboxylate groups located in the central membrane. When a maximal water permeation is observed, a tilted structure allows a toroidal pore formation that we observed transiently in the simulations (Figure 1b). An idealized toroidal pore is shown in Figure 1b, maximizing favorable interactions with the environment for both water interacting with polar and membrane interacting with lipophilic moieties. [30,32] The water transport through unimolecular channels it is of outmost importance as proved by the woks of Hou et al. [18][19][20] However when the channels are unfavorably oriented in the membrane aggregated molecular self-assemblies may be considered. New mechanistic concepts can be used to describe the formation of multivalent pores. So we consider herein an innovative multivalent combinatorial strategy supposing that the exceptional channel PA platforms A-D combining variable pore dimensions and charged negative carboxylate or positive amino groups on their peripheries may allow: a) the simultaneous steric restriction, a key feature influencing channel selectivity and b) the charge driven enhanced water transport allowing water molecules to permeate the toroidal channels by mimicking the key functions of AQPs.

The lipid used for the water transport assays consists of egg yolk L-α-phosphatidylcholine (EYPC) used to prepare Large Unilamellar Vesicles (LUV) of 100 nm in diameter, maintaining the lipid background permeability of ~50 µm/s at 20 °C. The functional pillar [5]arene A-D channels were combined and incorporated into this pre-formed vesicle suspension at different molar compound to lipid ratios (0.04%, 0.09%, 0.18%, 0.37%, 0.75%, 1.5% mCLRs) in which channels were added by addition of dimethylsulfoxide (DMSO) solutions. The water transport water molecules/s/channel, respectively and they can be detected at higher mCLR = 1.5 % (Figure 2). Such a difference in performances explain that bis carboxylate (C) and rigid alkene (D) functionalized compounds are better fixed/oriented within membranes when compared with the most flexible compounds A and B. Several compounds or their combinations show a drop in overall activity at higher membrane loadings because of poor solubility or membrane partition, which might occur if higher order aggregates are formed at concentrations above 1 mM. Overall, the rim-differentiated and symmetrical carboxylic and ammonium pillar [5]arenes A-D and their mixtures, can be assembled into artificial channels suitable to transport water and protons. Their combinatorial self-assembly lead to synergic increase of water permeabilities, while preventing the translocation of hydrated alkali cations. Slight modifications to the number and disposition of rim carboxylic and ammonium functional groups surrounding the hydrophilic entries of the PA channels can drastically change the water permeability. These channels are capable of transporting water at a rate that is one order of magnitude below the natural AQP. More importantly, salt rejection and their extremely low proton transport offer the closest functional mimic of the natural AQP through a synthetic scaffold. The present studies are of tremendous importance showing adaptive performances of mixtures combining simultaneous steric restriction and the charge driven enhanced water transport. Our findings show that the Artificial Water Channels story may reserve novel surprises, especially when channels are constructed from libraries with a multitude of members. The present study revealed a new paradigm: compounds may exhibit a dominant synergistic water transport activity in the presence of other co-active components. This contribution adds several new behaviors to the systematic rationalization of active artificial water channels mimicking the key functions of AQPs (Figure 2), towards the construction of industrial relevant membranes for desalination. [33]

Figure 1 .

 1 Figure 1. a) Structures of rim-differentiated and symmetrical pillar[5]arenes A-D. b) The self-assembly of PA channels via hydrogen bonding and charge interactions, resulting in the formation of combinatorial libraries of water channel architectures and water channels formation of aggregated PAs A2 dimers resulted for molecular simulation (reproduced from reference [30]) and resulting in the possible formation of toroidal pores of charged (COO -and Et2NH + ) inner hydrophilic surfaces when inserted in the bilayer membranes

  measurements through these channels were obtained by stopped-flow light-scattering experiments. Under hypertonic conditions driven by outwardly 10 mM phosphate buffer solution and 100 mM KCl osmotic gradient, the shrinkage of the liposomes increased the light-scattering signal. The kinetics of the vesicle shrinkage indicated that PA channels' permeability increases when mCLRs increases. The net permeability (Pw) and single-channel permeability (Ps) for the single compounds present a maximum of Pw=19.1 μm/s and of Ps= 1.4•10 8 water molecules/s/channel (mCLR = 0.37 %) for compound C and of Pw=11.6 μm/s and of Ps= 3.54•10 7 water molecules/s/channel (mCLR = 0.75 %) for compound D which are the most permeable towards water and which are only one order of magnitude lower than that of AQP, whereas for A, and B, the maxima values are lower: 4.8 μm/s and of Ps= 1.14•10 7 water molecules/s/channel and 5.4 μm/s and of Ps= 7.4•10 6

Figure 2 .

 2 Figure 2. Water transport activity of PA channels and mixtures in liposomes. The water permeabilities of the liposomes containing different mCLRs of compounds A, B, C D, and their combinations measured under hypertonic conditions from the post-mix assays. The liposomes (0.025 mg/mL of EYPC) were abruptly exposed to a hypertonic buffer solution of 100 mM KCl in PBS (10 mM, pH 6.4) at 20°C from the post-mix experiments.

Acknowledgements

This project has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement no 860592.

Keywords: artificial water channels • pillar[5]arene • selfassembly • dynamic combinatorial libraries • bilayer membranes

Notes:

The authors declare no competing financial interest.


Zhujie He

Huanfeng Huang

Zhuodi Chen

Yuqian Liang

Zhixiang Huang

Shunlian Ning

Lilin Tan

Mihail Barboiu

Dawei Wang

Cheng-Yong Su

Shunnian Ning

A Lehn

TiH 2 -supported Ru catalyst with unusual electron transfer behaviour for highly efficient carbon dioxide methanation at low temperature

Introduction

The excess emission of carbon dioxide (CO 2 ), the primary greenhouse gas, have caused challenging issues including global warming, biological system imbalances, and ocean acidification in the past decades. 1,2 It is therefore highly desirable to develop capable technology and devices for efficient capture, utilization, and storage of CO 2 . As a promising strategy to achieve net-zero emission fuel cycle, catalytic CO 2 hydrogenation have attracted extensive interest, since it allows the conversion of CO 2 into various value-added chemicals such as CO, CH 4 , CH 3 OH, HCOOH, and other hydrocarbons. 2,3 The catalytic hydrogeneration of CO 2 to produce CH 4 (known as CO 2 methanation or Sabatier reaction) are of particular significance and growing interest in recent years, which has found applications in several important industrial fields including the production of synthetic nature gas and the hydrogen feedstock purification for ammonia production. 4,5 Due to the strong C=O double bonds of CO 2 (bond energy of 750 kJ mol -1 ), 6 CO 2 methanation process is usually operated at high temperature (>300 °C) and in the presence of heterogeneous catalysts. Typical heterogeneous catalysts for CO 2 methanation are highly active metal nanoparticles (NPs; such as Ru, Rh, Co, and Ni [7][8][9][10][11][12][13][14] ) supported on reducible oxide supports (like TiO 2 , CeO 2 , and ZrO 2 7,8,13,15,16 ) that anchor metallic NPs and prevent NPs from agglomeration at high reaction temperature. For such heterogeneous catalysts, the metal-support interaction (MSI) has a critical impact on the physiochemical properties of catalysts and the catalytic hydrogeneration process. 17,18 Rational MSI regulation thus has been widely used as a predominant strategy to achieve excellent catalytic performance (activity, selectivity, and stability) for CO 2 hydrogeneration.

Typical MSI operating in catalytic CO 2 hydrogeneration includes electron transfer, strong metal-support interaction (SMSI), and hydrogen spillover. 7 Taking supported Ru catalyst, one of the most efficient heterogeneous catalysts towards CO 2 hydrogeneration, for an example, recent studies reveal that the support-metal electron transfer plays a crucial role in the product selectivity, by impacting on the adsorption and activation of CO intermediates. 16,19,20 Due to the partially positive charge on the C atoms of C=O bonds in CO intermediates, electron transfer from Ru to TiO 2 decreases the electron density around Ru NPs and thus moderately weakens the adsorption and subsequent activation of CO intermediates on Ru, leading to high CO selectivity over CH 4 . 20 In contrast, electron transfer from CeO 2 support to Ru increases the electron density around Ru NPs, which facilitates the adsorption and activation of CO intermediates and in turn results in 100% CH 4 selectivity. 16 SMSI is usually observed when reducible oxides (e.g., TiO 2 ) are used as support for metal NPs and (partially) reduced at high temperature (300-700 °C) under reducing atmosphere (typically, H 2 ) or ultrahigh vacuum, leading to the migration of support to metal NP surface and the subsequent formation of an oxide overlayer around metal NPs. 9,21 For TiO 2 -supported Ru catalysts, SMSI generally results in deteriorative activity but enhanced stability of Ru NPs due to the coverage of their surface sites by TiO 2 overlayer. 7 Moreover, due to the higher Fermi lever of partially reduced TiO 2 support as compared with Ru NPs, SMSI induces electron transfer from TiO 2 support to Ru NPs and thus results in high CH 4 selectivity, 7,22,23 in sharp contrast to TiO 2 -supported Ru catalysts prepared under mild conditions (e.g., wet impregnation method), in which the electron transfer from Ru to TiO 2 leads to high CO selectivity. 20 Hydrogen spillover also readily occurs when reducible oxide supported metal NP catalysts are used for CO 2 hydrogenation. Hydrogen spillover benefits the activation of H 2 , since abundant, highly active hydrogen atoms can be generated by the dissociative adsorption of H 2 on metal NP surfaces and the spontaneous migration of hydrogen atoms to the surface of reducible oxide support. 21 Therefore, hydrogen spillover has been been utilized to improve the activity of supported catalysts towards CO 2 hydrogenation. However, recent study based on CeO 2 -supported Ru catalysts reveals that hydrogen spillover should be regulated at an appropriate level to suppress the adverse hydration of catalyst surfaces, and also to achieve a balance with SMSI due to their competitive relationship. 16 Given the critical and complex roles of MSI in the performances of supported catalysts, the rational regulation of MSI is therefore of notable significance for highly efficient CO 2 methanation, which remains very challenging despite recent progresses. 17,24 Here, we develop a new heterogeneous catalyst for CO 2 methanation, by utilizing TiH 2 as the support for Ru NPs. TiH 2supported Ru NPs (denoted as mRTH, in which m refers to the mass loading of RuCl 3 precursor in mg) can be readily prepared in solution by the deposition of Ru NPs on TiH 2 . Benefitting from the highly active, negatively charged hydrogen species of H -in TiH 2 , spontaneous oxidation of TiH 2 surface occurs under ambient conditions, 25 leading to the formation of a thin surface layer of oxides and oxyhydrides (denoted as TiO x H y ) between bulk TiH 2 and Ru NPs. The TiO x H y surface layer acts as partially reduced oxide support for Ru NPs and enables notable electron transfer from support to Ru NPs, which thus remarkably enhances the activation of CO intermediates on Ru NPs, and in turn results in superior activity and CH 4 selectivity towards catalytic CO 2 methanation. Despite similar electron transfer behaviour observed in SMSI-mediated TiO 2 -supported Ru catalysts, the TiH 2 -supported Ru NPs do not require reduction treatment at very high temperature (300-700 °C), and thus avoids the formation of adverse oxide overlayer on Ru NPs that impairs the catalytic activity and/or selectivity of Ru NPs. Moreover, such electron transfer behaviour has not been observed in traditional TiO 2 -supported Ru catalysts synthesized under similar mild conditions, in which the electron transfer from Ru to TiO 2 results in high CO selectivity. Additionally, the electron transfer is appropriately coupled with hydrogen spillover effect to achieve synergistic balance between CO activation, H 2 activation and catalyst surface hydration, which finally imparts superior activity and CH 4 selectivity to TiH 2supported Ru NPs for catalytic CO 2 methanation.

Experimental Chemicals

Ruthenium trichloride (RuCl 3 ) was purchased from Bide Pharmatech Ltd. Titanium hydride (TiH 2 , 95%; >99% Ti) was purchased from Strem Chemicals Inc. Ethyl Glycol (99.5%) was purchased from Titan Scientific Company Ltd, Shanghai. Millipore deionized water (18.2 MΩ‧cm) was used for solution preparation. All reagents were used as received unless otherwise noted.

Synthesis of mRTH (m = 50, 100, 150, and 200) TiH 2 powder (200 mg) was added to a round bottom flask (100 mL) with ethylene glycol (EG; 25 mL) and deionized water (25 mL), and treated with ultrasonication and vigorous stirring for 15 min. Then RuCl 3 (50 mg) were added to the solution, and treated with ultrasonication and vigorous stirring for 60 min. Subsequently, the obtained brownish-black solution was heated at 150 °C with an oil bath for 180 min, and allowed to cool down to room temperature under ambient condition. The product, denoted as 50RTH, was collected by centrifugation and washed with water and ethanol three times. The synthesis of other samples of 100RTH, 150RTH, and 200RTH was achieved by changing the mass of RuCl 3 while keeping all the other parameters unchanged.

Synthesis of Ru NPs

The synthesis of Ru NPs (as control sample) was accomplished by following the same procedure for the synthesis of Ru NPs described above. Briefly, RuCl3 (50 mg) were added to a round bottom flask (100mL) with EG (25 mL) and deionized water (25 mL) under 15 min of ultrasonication and vigorous stirring, and heated at 150 °C with an oil bath for 180 min. After cooling down the solution to room temperature, the product was collected by centrifugation and washed three times with water and ethanol.

Characterization

The powder X-ray diffraction (PXRD) patterns were collected with a Rigaku MiniFlex 600 X-ray diffractometer (Cu Kα). The morphology of samples was recorded with a field-emission scanning electron microscope (FE-SEM, Hitachi, SU8010) operated at 5 kV and 10 mA. The transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) images and energy-dispersive X-ray spectroscopy (EDX) data were recorded with a spherical aberration corrected transmission electron microscope (SAC-TEM, JEM-ARM200P) operated at 200 kV. The chemical states of elements were probed by X-ray photoelectron spectroscopy (XPS, Thermo Fisher Nexsa). The surface profile and potential were obtained with an atomic force microscope (AFM) and Kelvin probe force microscope (KPFM; Bruker Dimension Fastscan). The precise element composition was detected with an inductively coupled plasma-Please do not adjust margins Please do not adjust margins emission spectrometer (ICP-OES) equipped with an Agilent 5110 spectrometer. Diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) were conducted on a Thermo Scientific Nicolet iS20 spectrometer.

The hydrogen spillover effect was investigated by temperature-programmed reduction (TPR) with a chemisorption analyser (BELCAT II, MicrotracBEL). Before TPR test, samples (51 mg) were heated to 100 °C at a ramp rate of 10 °C min -1 , kept at 100 °C for 30 min to remove adsorbed H 2 O, and cooled to 50 °C under nitrogen atmosphere. Then the samples were kept for 60 min in 10% H 2 -90% N 2 flow at a flow rate of 30 mL min -1 . Finally, the samples were heated to 900 °C at a ramp rate of 5 °C min -1 in a 10% H 2 -90% N 2 flow at a flow rate of 30 mL min -1 .

Catalytic CO 2 hydrogenation

The catalytic performances of mRTH for CO 2 hydrogenation were evaluated with a commercial evaluation system (CEL-HPR100T+, Beijing China Education Au-Light Co., Ltd). Typically, catalyst powder (40 mg) was placed in a quartz cup (150 mL) that was sealed within stainless steel and transferred into an autoclave. A CO 2 /H 2 mixture (CO 2 :H 2 = 1:4; 4 bar) was filled in the autoclave after evacuation for several times, and allowed to proceed at 200 °C. The products were detected with a gas chromatography (FL 9790) equipped with a flame ionization detector and thermal conductivity detector.

Results and discussion

mRTH were prepared by a wet synthesis procedure, using EG as reductant (cf. Experimental for details). As presented in the typical SEM and TEM images in Fig. 1a and S1, TiH 2 are large particles with size of around 2-10 μm, which show a lattice spacing of 2.58 Å corresponding to the (111) lattice plane of cubic TiH 2 . 25,[27][28][29] . Each microscale TiH 2 particle is coated with Ru NPs of around 3 nm in average diameter, to form mRTH with hybrid structure (Fig. 1b-d). Interesting, the Ru NPs exist in the form of large aggregates of 30-100 nm in diameter (Fig. 1c & Fig. 2a-g), which is likely due to the aggregation of adjacent Ru NPs induced by van der Waals interactions. 30,31 Each small Ru NP in large Ru aggregates can be identified in HRTEM image (Fig. 1d), which shows a lattice spacing of 2.03 Å corresponding to the (101) plane of Ru with hexagonal crystalline structures. 32 The EDX element maps of mRTH (m = 100 in Fig. 1e; m = 50, 150, and 200 in Fig. 2h-j, respectively) confirm the elemental composition of Ru and Ti in the samples, and the precise content of Ru and Ti is determined by ICP-OES (Table S1), which gives a a Ru loading of 5.7, 9.8, 15.5, and 17.3 wt % in 50-, 100-, 150-, and 200RTH, respectively. The appearance of O in the EDX elemental maps should be attributed to the formation of TiO x H y layer on the surface of TiH 2 , which is induced by the oxidation of TiH 2 by atmospheric oxygen under ambient conditions. 33,34 The PXRD patterns of mRTH show a set of peaks centred at 34.9, 40.5, 58.6, 70.1, and 73.7°, respectively, which is indexed to cubic TiH 2 (PDF #07-0370). By contrast, the PXRD peaks of hexagonal Ru (PDF #06-0663) located at 44.0° are only visible in the mRTH samples with high Ru loading in 100-, 150-, and 200RTH; the PXRD peaks of Ru in 50RTH samples is almost invisible, likely due to the low loading of Ru NPs with small size (ca. 3 nm). [35][36][37][38] The PXRD patterns, together with the TEM images and EDX element maps, thus collectively verify the hybrid Ru/TiH 2 structure of mRTH.

It is worth noting that the spatial dispersion of Ru NPs in mRTH relates closely to Ru loading (Fig. 2). As the Ru loading increases, Ru NPs organize into discrete aggregates in 50RTH (5. The MSI between Ru NPs and TiH 2 were systematically investigated by AFM coupled with KPFM, XPS, and H 2 -TPR. AFM-KPFM was first used to investigate the charge transfer behaviour in mRTH. By selecting single particle of TiH 2 and mRTH (Fig. 3a-e), the typical surface potential of TiH 2 and mRTH can be obtained and compared by contact potential difference (CPD) given by CPD = E sample -E substrate , where E sample and E substrate are the measured surface potential of sample and substrate, respectively, and n-Si(111) was used as the substrate for our KPFM measurements (Fig. 3f-j and Table S2). 39,40 The CPD shows a positive corelation with the Ru loading in sample, increasing from -8 mV for TiH 2 , to 2 mV for 50RTH, 26 mV for 100RTH, 37.4 mV for 150RTH, and 68.8 mV for 200RTH. The CPD shift from negative (TiH 2 ) to positive (mRTH) values confirms the electron transfer from TiH 2 to Ru NPs. 41 Moreover, due to the positive correlation between CPD and Fermi level, 50RTH with the lowest positive CPD should have the lowest Fermi level among all mRTH, which thus should allow for the easiest electron transfer from TiH 2 to Ru NPs. 40 To obtain more details about the interfacial electron transfer and the chemical states of interface species in mRTH, XPS spectra were collected. For TiH 2 and mRTH, the high-resolution XPS spectra of Ti 2p 3/2 and Ru 3d 5/2 indicate that in all samples the surface Ti species exist in the forms of Ti 4+ and defective TiO 2-x in the TiO x H y surface layer (Fig. 4a; cf. Table S3 for the binding energy corresponding to each peak), and Ru exists in the form of Ru 0 and Ru 4+ (Fig. 4b). 27,28,33,34,42,43 The formation of TiO 2-x and Ru 4+ is induced by the spontaneous oxidation of TiH 2 and Ru surfaces by atmospheric oxygen under ambient conditions, 7,34,43,44 which agrees well with the results of EDX element maps shown in Fig. 1e. Moreover, as compared with TiH 2 , the mRTH samples show increasing binding energies of Ti 4+ and defective TiO 2-x , and the binding energy shifts increase steadily from 50RTH to 200RTH. In contrast, the mRTH samples present decreasing binding energies of Ru 0 and Ru 4+ as compared with Ru NPs, and the absolute binding energy shifts decrease steadily from 50RTH to 200RTH (Fig. 4b). Such opposite binding energy shift trends in TiH 2 and Ru NPs imply interfacial electron transfer from TiO x H y to Ru, which agrees well with our KPFM results (Fig. 3) and thus confirms the MSI in mRTH. Notably, the interfacial electron transfer from TiO x H y to Ru results in the formation of electron-enriched Ru species (Ru δ-) in mRTH. As demonstrated later in this work, the Ru species are the dominate sites for CO 2 absorption; moreover, electronenriched Ru species could benefit the chemisorption of CO 2 and the formation of [O=C=O] δ-intermediates, and thus efficiently accelerate the CO 2 methanation reaction. It is worth noting that Please do not adjust margins

Please do not adjust margins the interfacial electron transfer from TiO 2 to metal NPs (e.g., Ru, 7 Pt, 25 and Ni 45 ) typically occurs in the case of SMSI, which is induced by the partial reduction of TiO 2 support when treated at high-temperature (typically, >300 °C) and under H 2 atmospheres. In contrast, for the TiO 2 -supported metal NPs (e.g., Ru 20 and Au 46 ) prepared under mild conditions that disfavour support reduction, the electron transfer usually occurs in opposite direction, that is, from metal NPs to TiO 2 . Therefore, the unusual charge transfer from TiO x H y to Ru NPs in our mRTH prepared under mild condition (wet synthetic process; see details in Experimental) should be attributed to the highly active, negatively charged H -in TiH 2 , which leads to the spontaneous oxidation of TiH 2 surface into TiO x H y layer consisting of oxides and oxyhydrides (cf. Ref. 23 and also the Ti 2p 3/2 and O 1s XPS spectra in Fig. 4), and thus enables the TiO x H y surface layer to acts as partially reduced oxide support for Ru NPs, similar to the role of TiO 2 partially reduced at hightemperature and under H 2 atmospheres in the case of SMSI.

To rationalize the opposite impact of Ru loading on the peak shifts in the Ti 2p 3/2 and Ru 3d 5/2 XPS spectra of mRTH, the spatial dispersion of Ru NPs on TiH 2 has to be taken into account. Note that the XPS spectra are collected based on a typical spot size of 30-500 μm, much larger than the size of all mRTH samples (< 5 μm; cf. Fig. 12), the XPS peak position could be regarded as a rough indicator of the binding energy averaged over all the surface Ti or Ru species, in which the surface Ti species could be estimated to be the same for all mRTH and the surface Ru species reply specifically on the Ru loading in each mRTH. Generally, increasing Ru loading leads to raised surface density of Ru NP aggregates that interact with the surface layer of TiH 2 , and accordingly enhanced overall MSI at the interface. In such a way, the MSI shows increasing impact on the binding energy of the surface Ti species of TiH 2 (approximately the same for all mRTH), and thus the Ti 2p 3/2 XPS peaks continuously shift upwards as the the Ru loading in 50-, 100-, 150-and 200RTH steadily increases (Fig. 4a). Notably, 100RTH presents much more remarkable shift of the Ti 2p 3/2 XPS peak position than 50RTH, which could be attributed to the much greater surface density of Ru NP aggregates in 100RTH (9.8 wt %; vs. 5.7 wt % in 50RTH). Moreover, the Ru NP aggregates in the second layer of 150-and 200RTH are in indirect contact with the surface layer of TiH 2 , and thus are less efficiently involved in MSI as compared with the Ru NP aggregates in the first layer that are in direct contact with the surface layer of TiH 2 . Although the overall MSI shows a rising trend as the Ru loading in 100-, 150-and 200RTH steadily increases, the overall MSI averaged over all the surface Ru species are declining, thus having a decreasing impact on the binding energies of surface Ru species and leading to downward shifts in the Ru 3d 5/2 XPS peak position (Fig. 4b). Moreover, in the case of 50-and 100RTH with all the Ru NP aggregates in direct contact with the surface layer of TiH 2 , the notable shift in the Ru 3d 5/2 XPS peak of 50RTH is likely due to the smaller size of Ru NP aggregates in 50RTH (38 nm; vs. 52 nm for 100RTH), which results in much stronger MSI and consequently much greater impact on the binding energies of Ru species in 50RTH.

The O 1s spectra of mRTH and TiH 2 are also analysed. As shown in Fig. 4c, all the O 1s spectra show three types of O species that correspond to the lattice O in TiO x H y and RuO 2 (denoted as O1), the O at defect (oxygen vacancy) sites or Ti-OH (O2), and the O in chemically adsorbed oxygen-containing species like H 2 O (O3). 25,33 The appearance of O1 and O2 peaks confirms the partial oxidation of surface TiH 2 resulted from the highly active nature of the negatively charged H -in TiH 2 . Moreover, the Ru loading in mRTH correlates with greater integrated peak areas of O2 and O3, which is likely due to the low migration energy of H -at the Ru-metal hydride interface. 26 Briefly, the low migration energy favors the migration of H - from TiH 2 to the Ru, and higher Ru loading thus allows for the formation of more oxides and oxyhydrides of Ti through ambient oxidation of H -at the interface, and gives rise to the increased peak areas of O2. Both the oxygen vacancy and OH group in TiO x H y surface layer have good binding affinity to H 2 O under ambient conditions, 47,48 thus leading to enhanced adsorption of H 2 O and increased peak area of O3 at higher Ru loading level.

H 2 -TPR was also used to further evaluate the Ru-TiH 2 MSI as well as the reducibility of TiH 2 support. 49 As presented in Fig. 4d, all the H 2 -TPR profiles of TiH 2 and mRTH show two H 2 consumption peaks in the temperature range of 250-750 °C, one centred in the range of 400-450 °C corresponding to the hydrogen storage by β-TiH x phase in TiH 2 , and the other centred at 580 °C corresponding to the hydrogen storage by α-Ti phase in TiH 2 . [50][51][52][53] The later peaks locate at almost the same temperature for TiH 2 and mRTH, in agreement with the results reported in literature. 25 In contrast, the former peaks related to the reduction of TiO x H y surface layer and bulk TiH 2 shift towards lower temperature when the Ru loading in mRTH increases, indicating improved reducibility of TiH 2 by Ru loading. The improved reducibility of TiH 2 could be interpreted as a consequence of Ru-TiH 2 MSI. Specifically, as implied by the XPS Please do not adjust margins Please do not adjust margins spectra in Fig. 4a-c, the charge transfer from TiH 2 to Ru increases the electron density at the Ru-TiH 2 interface, and thus enables the surface TiO x H y layer to be partially reduced at a lower temperature. Moreover, H 2 could be remarkably activated by TiO x H y -supported Ru NPs through hydrogen spillover effect [54][55][56] , and oxygen vacancies on TiO x H y -support could further enhance the hydrogen spillover effect by facilitating the transfer of active hydrogen species, 57 thus further enhancing the reducibility of TiH 2 . As both the Ti-Ru charge transfer behaviour and the hydrogen spillover effect are positively related to the Ru loading (abundant active hydrogen species can be generated by more Ru NPs), 200RTH with the highest Ru loading shows notable decrease, from 469.7 to 401.1 °C, in the peak temperature for the reduction of TiH 2 .

The catalytic performances of mRTH towards CO 2 methanation were evaluated at 200 °C and in an autoclave filled with 4 bar of reaction gas of stoichiometric ratio (20 vol.% CO 2 and 80 vol.% H 2 ). Control experiments using TiH 2 support and Ru NPs as catalysts were also conducted under otherwise identical conditions. Approximately, the conversion of CO 2 with representative catalysts (TiH 2 , Ru NPs, and 100RTH; Fig. 5a) increases linearly with reaction time, which shows a typical feature of heterogeneous catalysis occurring on the catalyst surface and implies a reaction kinetics depending primarily on the surface properties of catalysts. An overall comparison of CO 2 conversion and CH 4 selectivity between TiH 2 , Ru NPs, and mRTH are presented in Fig. 5b. Despite notable difference in CO 2 conversion, all the TiH 2 , Ru NPs, and mRTH show superior CH 4 selectivity of >99.4%. Moreover, all mRTH show remarkably improved conversion (44.7, 86.5, 72.8, and 61.0% for 50-, 100-, 150-, and 200RTH, respectively) as compared with Ru NPs (29.0%) and TiH 2 (1.2%), which thus indicates notable synergistic effect between TiH 2 and Ru. The superior selectivity and activity towards CO 2 methanation demonstrates the advantages of mRTH over traditional Ru-TiO 2 heterogeneous catalyst prepared under similar mild conditions and SMSImediated Ru-TiO 2 heterogeneous catalysts synthesized by high-temperature reduction. 8,20 As compared with the former one, mRTH show superior selectivity and activity towards CH 4 production, resulting from the usual electron transfer from TiO x H y surface layer to Ru NPs that leads to the generation of electron-enriched Ru NPs with enhanced activation capability for CO intermediates. As compared with the later one, the superior activity of mRTH could be attributed to the efficiently exposed Ru NPs, whose activity are otherwise largely impaired due to the coverage of adverse oxide overlayers in SMSImediated Ru-TiO 2 heterogeneous catalysts.

A more accurate comparison of the catalytic activities between TiH 2 , Ru NPs, and mRTH was made based on specific activities, which are indicated by the reaction rate of CH 4 with respect to the total catalyst mass (denoted as r(CH 4 , Cat.)) or the mass of Ru in each catalyst (denoted as r(CH 4 , Ru)), respectively (Fig. 5c). Given the same mass of catalysts used in CO 2 methanation, the trend in r(CH 4 , Cat.) is, as expected, in line Please do not adjust margins Please do not adjust margins with the trend in CO 2 conversion. However, r(CH 4 , Ru) shows two different features: (1) impressively, 50-, 100, 150-, and 200RTH delivers a large r(CH 4 , Ru) of 167.8, 168.7, 81.2 and 54.8 mmol h -1 g Ru -1 , which is 21.0, 21.1, 9.6, and 6.2 times higher than that of Ru NPs (7.6 mmol h -1 g Ru -1 ), respectively, and thus implies that the catalytic activity of Ru NPs can be remarkably enhanced by TiH 2 support; (2) 50-and 100RTH with low loading of Ru NP aggregates in direct contact with TiH 2 surface show very comparable r(CH 4 , Ru) of 167.8 and 168.7 mmol h -1 g Ru -1 , respectively, which are both much higher than 150-and 200 RTH with high loading of Ru NP aggregates in both direct and indirect contact with TiH 2 surface. The two features of r(CH 4 , Ru) clearly indicate that the Ru-TiH 2 interface could substantially increase the catalytic activity of Ru, and direct Ru-TiH 2 contact induces much more efficient interface for boosting the catalytic activity of Ru than indirect Ru-TiH 2 contact. These results thus confirm and highlight the critical role of appropriate Ru-TiH 2 MSI on the catalytic activity of mRTH towards CO 2 methanation, which will be discussed in detail later in this work. Moreover, despite comparable specific activity of Ru NPs in 100RTH and 50RTH, 100RTH is selected as the optimum catalyst for CO 2 methanation, due to its higher Ru loading that benefits more efficient utilization of TiH 2 (indicated by higher r(CH 4 , Cat.)). It is also worth noting that catalytic activity of 100RTH, indicated by its r(CH 4 , Ru) up to 168.7 mmol h -1 g Ru -1 , is superior to most of the Ru-based heterogeneous catalysts reported in literature, and thus enables 100RTH to be among the best Ru-based heterogeneous catalysts for CO 2 methanation, particular those working efficiently at temperature ≤200 °C (cf. Table S4).

The stability of mRTH was also evaluated. Taking 100RTH for instance, the CO 2 conversion and CH 4 selectivity show neglectable change even after 5 cycles (equivalently, 25 h; Fig. 5d) of successive operation, which thus demonstrates its excellent cycling stability.

To get insights into the excellent catalytic activity of 100RTH and the activity differences between various mRTH, two important control experiments were adequately designed and conducted, and TiH 2 , Ru, and 100RTH were selected as representative catalysts. The first one was the sorption of CO 2 on catalyst surfaces, in which CO 2 was first allowed to be adsorbed on catalyst surface at 30 °C for 30 min, and then desorbed at elevated temperature (5 °C min -1 ) in N 2 flow (50 mL min -1 ). The whole sorption processes were monitored with in situ DRIFTS. As shown in Fig. 6a-c, the DRIFTS spectra of TiH 2 , Ru, and 100RTH all show absorption peaks at 1640 cm -1 , which is attributed to the adsorbed CO 2 species of [O=C=O] δ-on catalyst surface. [60][61][62] However, the sorption peaks at 1640 cm -1 in the spectra of Ru NPs and 100RTH are much more intense than the corresponding peaks of TiH 2 at the same temperature. Moreover, another sorption peaks can also be found at ca. 2100 cm -1 in the enlarged DRIFTS spectra of Ru NPs and 100RTH, which correspond to Ru(CO) n species generated by the partial dissociation of adsorbed CO 2 on Ru NP surfaces. 7,63,64 The peaks of Ru(CO) n species, however, are not found in the spectra of TiH Please do not adjust margins Please do not adjust margins roughly remain their intensity; in sharp contrast, the Ru(CO) n peaks of 100RTH decline rapidly in their intensity and even vanishes after keeping 100RTH at 200 °C for 50 min. As revealed below, hydrogen stored in TiH 2 can be released at elevated temperature (Fig. 6d). The rapid declined peak intensity of Ru(CO) n in 100RTH thus should be attributed to the reduction of adsorbed CO species by hydrogen released from TiH 2 . 7 The second control experiment is the CO 2 methanation in the absence of external H 2 source and under otherwise identical conditions. As expected, despite the high intrinsic activity of RU NPs towards catalytic CO 2 hydrogenation, 65 no product is detected for Ru NPs due to absence of hydrogen (Fig. 6d). In contrast, TiH 2 shows a high H 2 production rate of 298.1 μmol h - 1 g cat. -1 , which is due to the release of hydrogen stored in TiH 2 and thus verifies the hydrogen storage and release capability of TiH 2 . 28,51 This explains why CO 2 can be reduced by TiH 2 even in the absence of external H 2 source. However, the negligible production rate of CH 4 and low selectivity of CH 4 over CO (denoted by the CH 4 /CO rate ratio) indicate the poor intrinsic activity of TiH 2 towards catalytic CO 2 methanation. As compared with TiH 2 , 100RTH delivers 22 times higher CH 4 production rate and 1.9 times higher CH 4 selectivity. These results, together with the results shown in Fig. 6a-c, collectively suggest that the Ru NPs in 100RTH are not only the predominant sites for CO 2 adsorption but also the predominant catalytic sites for CO 2 methanation. We further note that the CH 4 selectivity of 100RTH in the absence of H 2 is much worse than that in presence of stoichiometric amount of H 2 (Fig. 6a), which should be to the limited amount and very low partial pressure of H 2 released from TiH 2 . 66 Based on all the above discussion, we propose a plausible relationship between structure, MSI and catalytic performance of mRTH (Scheme 1). As well demonstrated in literature, the activation of carbonyl and hydrogen plays a critical role in efficient CO 2 methanation. 7,8,16 The carbonyl with partially positively charged C atoms in CO 2 and Ru(CO) n intermediates can be activated by directing electron transfer from support to Ru NPs, which is beneficial for carbonyl to get electrons from electron-enriched Ru NPs and thus to be easily stabilized for subsequent conversion into CH 4 . Moreover, although hydrogen activation can be readily achieved on Ru NPs through spillover effect, excess Ru NPs should be avoided, since intense hydrogen spillover effect can be induced, which inversely leads to deteriorative catalytic activity towards CO 2 methanation by strengthening the adsorption of H 2 O molecules and hindering the removal of H 2 O molecules from catalyst surfaces. 16 Accordingly, the interfacial charge transfer and Hydrogen spillover effect should be well regulated for optimal catalytic activity. In our mRTH, benefitting from the TiO x H y surface layer on TiH 2 and the direct Ru-TiO x H y contact in 50-and 100RTH, notable electron transfer from Ti to Ru can be achieved under mild synthetic condition (Fig. 4), which enriches the electron on Ru NPs and thus facilitate the carbonyl activation for efficient CO 2 methanation. Meanwhile, the low to moderate Ru loading in 50-and 100RTH allows favourable hydrogen spillover effect (positively corelated to the Ru loading) for efficient CO 2 methanation by hydrogen activation. Therefore, the electron transfer and hydrogen spillover effect in 50-and 100RTH collectively contribute to the superior catalytic activity towards efficient CO 2 methanation. By comparison, the higher loading Ru NPs with partially indirect Ru-TiO x H y contact in 150-and 200RTH induces less sufficient Ti-Ru electron transfer for carbonyl activation, and disadvantageous hydrogen spillover effect due to the enhanced adsorption of H 2 O molecules on catalyst surface, which in turn results in their much lower activity towards CO 2 methanation.

Conclusions

In conclusion, we have developed a new heterogeneous catalyst of TiH 2 -supported Ru NPs for highly efficient CO 2 methanation. Benefitting from the highly active, negatively charged hydrogen species of H -in TiH 2 , the TiH 2 -supported Ru NPs show usual charge transfer from support to Ru NPs, which has not been achieved in the traditional TiO 2 -supported Ru catalysts synthesized under similar mild conditions. Instead, TiH 2supported Ru NPs show charge transfer behaviour similar to that in SMSI-mediated TiO 2 -Ru catalysts prepared under high temperature reduction conditions. Moreover, as compared with SMSI-mediated TiO 2 -Ru catalysts, the far milder synthetic condition for TiH 2 -supported Ru NPs avoids the formation of adverse oxide overlayer on Ru NPs, which leads to the remarkably enhanced activation of CO intermediates on the TiH 2 -supported, electron-enriched and efficiently exposed Ru NPs. Meanwhile, H 2 can also be activated by TiH 2 -supported Ru NPs via hydrogen spillover effect, inevitably accompanied by the adverse hydration of catalyst surface. The electron transfer behaviour and hydrogen spillover effect are readily regulated by tuning the Ru loading, and a moderate Ru loading of 9.8 wt.% in 100RTH is optimized, in which the impacts of Ru loading on CO intermediate activation, H 2 activation and catalyst surface hydration are synergistically balanced. When evaluated at a low reaction temperature of 200 °C and under 4 bar pressure, 100RTH is capable of delivering a notable CH 4 selectivity of 99.8% and a remarkable CH 4 production rate of 168.7 mmol h -1 g Ru -1 , which is 21.1 times more active than pure Ru NPs. Our work thus opens up a new avenue for the development of advanced Scheme 1. Proposed relationship between structure, MSI and catalytic performances of mRTH towards CO 2 methanation.

  7 wt %), a dense layer of Ru NP aggregates in 100RTH (9.8 wt %), a dense Ru NP aggregate layer with excess Ru aggregates in 150RTH (15.5 wt %), and dense double aggregate layers in 200RTH (17.3 wt %); meanwhile, the size of Ru NP aggregates in 100-, 150-and 200RTH is comparable (ca. 52 nm), while the size of Ru NP aggregates in 50RTH is smaller (ca. 38 nm) due to the low surface coverage of Ru NPs on TiH 2 (cf. Fig. 1b-d and Fig. 2b-g). Moreover, all the Ru NP aggregates are in direct contact with TiH 2 in 50-and 100RTH. In contrast, only the first Ru NP aggregate layer is in direct contact with TiH 2 , while the other Ru NP aggregates are separated by the first Ru layer in 150-and 200RTh. Such differences in the surface coverage, size, and spatial organization of Ru NP aggregates leads to the varied interfacial structure of mRTH, which in turn, as demonstrated later in this work, have critical impact on the MSI (interfacial

Fig. 1 (

 1 Fig. 1 (a) SEM image of TiH 2 . (b) SEM, (c) TEM, and (d) HRTEM images of 100RTH. (e) EDX element maps of 100RTH. (f) PXRD patterns of mRTH, TiH 2 and Ru NPs.

Fig. 2 (

 2 Fig. 2 (a) Schematic illustration of spatial dispersion of mRTH with increasing Ru loading. (b-d) SEM and (e-g) TEM images, and (h-j) EDX element maps of mRTH (m = 50, 150, and 200). cf. the data for 100RTH in Fig. 1b-e.

Fig. 3 (

 3 Fig. 3 (a-e) AFM height images and (f-j) the corresponding KPFM potential profiles of TiH 2 and mRTH. (a, f) TiH 2 , (b, g) 50RTH, (c, h) 100RTH, (d, i) 150RTH, and (e, j) 200RTH. CPD in (f-j) is estimated by the potential difference between site A (sample) and B (substrate).

Fig. 2 (

 2 Fig. 2 (a-c) High-resolution XPS spectra of Ru NPs, TiH 2 , and mRTH, (a) Ti 2p 3/2 , (b) Ru 3d 5/2 , and (c) O 1s. (d) H 2 -TPR profiles of TiH 2 and mRTH.

Fig. 3

 3 Fig. 3 Reaction kinetics of catalytic CO 2 methanation and catalyst stability. (a) Typical CO 2 conversion curves as a function of reaction time. (b) CO 2 conversion and CH 4 selectivity after 5 h of reaction. (c) Reaction rate of CH 4 with respect to catalyst mass and Ru mass in catalyst, (d) Catalyst stability of 100RTH.

  Fig. 4 (a-c) In situ DRIFTS spectra of CO 2 sorption on (a) Ru NPs, (b) TiH 2 , and (c) 100RTH at varied temperature. (d) Reaction rate of catalytic CO 2 methanation in the absence of external H 2 .

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Acknowledgements

This work was financially supported by the NSF (2021A1515010098), DST (STKJ202209075), CCE LAB (2111005) of Guangdong Province, the NKRD Program of China (2021YFA1500401), the NNSF of China (21821003, 21890380) and the LIRT Project of Guangdong PRTP (2017BT01C161).

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Conflicts of interest

There are no conflicts to declare. ToC Figure

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