Dr Serena Arnaboldi

Dr Gerardo Salinas

Prof. Dr Giorgia Bonetti

Prof Patrick Garrigue

Dr Roberto Cirilli

Tiziana Benincori

Dr Alexander Kuhn
email: kuhn@enscbp.fr

Autonomous Chiral Microswimmers with Self-mixing Capabilities for Highly Efficient Enantioselective Catalysis

Keywords: Electrosynthesis, inherently chiral oligomers, microswimmers, self-propulsion, heterogeneous catalysis

The development of chiral catalysts plays a very important role in various areas of chemical science. Heterogeneous catalysts have the general advantage of allowing a more straightforward separation from the products. One specific case of heterogeneous catalysis is electrocatalysis, being potentially a green chemistry approach. However, a typical drawback is that the redox conversion of molecules occurs only at the electrode/electrolyte interface, and not in the bulk of the electrolyte. The second limitation is that the electrodes have to be physically connected to a power supply to induce the desired reactions. To circumvent these problems, we propose here to replace macroscopic electrodes with an ensemble of self-propelled microswimmers, moving autonomously in solution while transforming simultaneously a prochiral starting compound into a specific enantiomer with a very high enantiomeric excess, accompanied by a significantly increased production rate of the favorite enantiomer.

Introduction

Chiral molecules manifest their peculiarities in different ways in nature. For example, the homochirality of compounds in biochemical systems is of outstanding importance in the pharmaceutical and agrochemical industry. Many enantiopure pharmaceutical compounds are obtained by separation methods, often involving chromatographic columns, or via catalysis. [1] An interesting approach is the use of heterogeneous catalysis to minimize solubility problems and allow a more straightforward purification of the obtained products. [2][3][4] Electroorganic synthesis employs electrons as reagents, making this methodology very attractive as a sustainable synthetic platform for organic redox reactions. [5][6][7][8] However, the success of electroconversion strongly depends on the efficiency of the selective induction of chirality, in synergy with optimized mass transport for a high overall yield. Chemically modified chiral electrodes are a useful tool for asymmetric synthesis, because they only require a small amount of chirality-inducing agents. Nevertheless, they often present as a major disadvantage the poor coverage of the surface with the corresponding chiral selector, thus leading to partially non-stereospecific transformations. An interesting alternative is the use of polymer-coated electrodes for chiral electroorganic synthesis. Abe et al. explored the use of a poly-ʟ-valine-coated graphite cathode for asymmetric electrochemical reductions in two consecutive reports. In the first communication, the asymmetric reduction of prochiral activated olefins was carried out, leading to optical yields of 25% and 43%, respectively. [9] In a second report, the same electrode was used for the asymmetric reduction of prochiral carbonyl compounds with an optical yield of 16.6%. [10] In another work, electrodes coated with a chiral poly-(N-substituted pyrrole)/palladium film, modified with L-(+)-lactic acid moieties as optically active groups, were used for the electrocatalytic hydrogenation of α-keto esters to produce the corresponding hydroxy esters with appreciable enantioselectivities. [11] However, a typical drawback in enantioselective electrocatalysis is that the redox conversion occurs only at the electrode/electrolyte interface, and not in the bulk of the electrolyte. In addition, the electrodes have to be physically connected to a power supply to induce the desired reactions. Herein, we propose to replace the classic macroscopic electrodes with an ensemble of self-propelled microswimmers.

The rational design of micro-and nano-swimmers has gained considerable attention during the last decade, [START_REF] Wang | Nanomachines: Fundamentals and Applications[END_REF][START_REF] Duan | [END_REF][14][15][16][17][18][19][20][21] due to the increasing number of potential applications in biomedicine, [22][23][24][25][26][27] environmental remediation, [28,29] (bio)sensing [30,31] and to mimic collective behavior of microorganisms. [32][33][34] The motion of these devices can be powered either by applying external stimuli (light, ultrasound, and magnetic or electric fields) or by the conversion of chemical energy into motion via different mechanisms, such as Marangoni effect, self-diffusiophoresis, selfelectrophoresis and bubble propulsion. [35][36][37][38][39][40][41][42][43][44][45] Recently, the use of self-propelled swimmers as microreactors, coupling the autonomous motion with the possibility to convert a substance in loco into a desired product, has been extensively studied. Such dynamic processes, also known as "chemistry on the fly", [46][47][48] are based on the synergy between an efficient propulsion and a specific surface functionalization, allowing these mobile platforms to move and actively mix reactants in the solution, combined with a high intrinsic surface reactivity towards the target molecules. This type of microswimmers opens completely new horizons in the frame of chemical synthesis, as they can be considered self-propelled microreactors. In recent years, such self-propelled swimmers for "chemistry-on-the-fly" have been introduced for the possible analysis and recognition of chiral information in solution. [49,50] Actually, scientists are aiming constantly for more innovative designs of these bioinspired objects, that can perform various tasks ranging from biosensing to drug delivery, driven by different mechanisms, including biochemical, optical, magnetic, and electric energy sources. For example, Escarpa's group designed an autonomous swimmer for "on-the-fly" biodetection of D-and L-amino acids, taking advantage of the dispersion of the enzymes D-or L-amino acid oxidase. [49] Recently, Muñoz et al. designed a self-propelled Ni@Pt micro-rocket, functionalized with fluorescent CdS quantum dots carrying a chiral host biomolecule, β-cyclodextrin (β-CD). [50] Such micro-rockets work as enantioselective supramolecular cargo platforms in aqueous solution. However, an ex-situ transduction mechanism (optical or electrochemical) is required in order to perform a quantitative analysis of chiral information in solution.

In this context, an interesting approach is to take advantage of the high enantioselectivity of inherently chiral thiophene-based compounds. In inherently chiral molecular materials, chirality and functional properties originate from the same structural element. The different diastereomeric interactions between the chiral surface and the molecular target in solution lead to thermodynamic differences, in terms of redox potentials, leading to the preferential formation of only one enantiomer. [51] The high specificity of such chiral selectors was exploited to develop e.g. a new approach based on bipolar electrochemistry for the absolute on-off recognition of enantiomers of a chiral probe. [52][53][54][55] Recently, these inherently chiral oligomers have also been used to design autonomous enzyme-based swimmers, able to produce enantioselective clockwise and anticlockwise motion as a function of the enantiomer present in solution. [56] In the present work, we employ these oligomers to design self-propelled asymmetric microswimmers, based on small pieces of zinc (Zn), partially functionalized with one of the antipodes of the inherently chiral material. Theoretically, in acidic media, the oxidation of Zn and the reduction of protons occur spontaneously with a standard redox potential difference of -0.76 V. Due to the large amount of electrons produced by this spontaneous reaction, a considerable fraction can be shuttled from Zn to the inherently chiral oligomer surface. Thus, in the presence of prochiral compounds, their asymmetric reduction allows the selective formation of only one enantiomer, mediated by the inherently chiral material that acts as a catalyst. In addition, the reduction of protons produces enough hydrogen (H2) on the swimmer surface to induce motion via a bubble propulsion mechanism. Such micro-engines offer considerable promise for accelerating the rate of asymmetric reactions with high yield through enhanced spontaneous micro-mixing, and thus should open up a wide range of practical applications.

Results and Discussion

Initial considerations

Due to their unique ability to selectively oxidize, at a specific applied voltage, the enantiomers of a chiral analyte, inherently chiral materials have gained attention in chiral bipolar electrochemistry for the development of on-off sensors to directly quantify the enantiomeric excesses of unbalanced mixtures of chiral probes. [52,54,55] Furthermore, this concept has been transferred to the field of self-propelled swimmers, to obtain a dynamic read-out of the configuration of chiral analytes through enantioselective trajectories. [52] However, the use of inherently chiral materials in asymmetric electrosynthesis remains a completely unexplored area, with an interesting application potential, considering their high enantiospecificity due to their ability to act as a catalyst to control the stereochemical outcome of the reaction. To study the beneficial effect of employing such inherently chiral materials for asymmetric electrosynthesis, the electroreduction of ketone functions (C = O) to alcohol (-OH) has been chosen as a model reaction. Commonly, the presence of highly reactive functional groups, such as phenyl or carboxyl groups, leads to undesired side products, making the electroreduction of ketones challenging. [57] Acetophenone (AP) and phenylglyoxylic acid (PGA) are suitable candidates since they can be reduced to 1-phenylethanol (1-PE) and mandelic acid (MA), respectively. Scheme 1. (A) Inherently chiral structures of the enantiopure antipodes of the (R)-BT2T4 and (S)-BT2T4 oligomers. (B) Schematic illustration of the experimental set-up used for the asymmetric synthesis together with the involved redox reactions.

Optimization of the enantioselective electrosynthesis

The enantiopure oligomers of an inherently chiral monomer (Scheme 1A, (R)-BT2T4 and (S)-BT2T4, respectively) were deposited galvanostatically on the surface of a macroscopic Zn wire, according to a previously described procedure. [52][53][54][55][56] These monomers, with 3,3'-bibenzothiophene as a central unit, have a well-defined electroactivity and can be easily electropolymerized. The two alpha homotopic positions on the thiophene wings guarantee the regioregularity of the oligomerization process. The high racemization barrier allows the separation of the enantiomers by high performance liquid chromatography (HPLC) [58] on a chiral stationary phase (CSP) under normal phase conditions. Both monomers are stable at room temperature and the chiral features can be fully transferred to the oligomers. [59] Scanning electron microscopy (SEM) analysis, in combination with energy dispersive X-ray spectrometry (EDS) (Figure 1), were used to evaluate the composition and morphology of a Zn wire, functionalized with one enantiomer of the BT2T4 oligomer. The deposit appears to be homogeneous with a globular morphology and a thickness of the order of 20 µm (Figure S1). The EDS signal shows that the oligomer modified part has as main components carbon, sulfur and oxygen (orange line), whereas only Zn was detected (grey line) on the pristine part (Figure 1). The possibility of using these modified chiral wires as a reactant for the autonomous stereoselective synthesis of 1-PE was evaluated by immersing 31 mm of the wire (30 mm of enantiopure oligomer modified Zn and 1 mm of pristine zinc) in an aqueous solution of 100 mM AP in 0.5 M H2SO4. The general experimental set-up, together with the mechanism of the redox reactions involved in the stereoselective synthesis is illustrated in Scheme 1B. As stated above, oxidation of Zn and reduction of protons take place spontaneously at the bare metal, whereas the stereospecific reduction of the prochiral precursor occurs on the part of the metal wire modified with the enantiopure oligomer. A certain amount of pristine Zn is required to be directly in contact with the solution in order to produce preferentially the necessary electron flow from the dissolving metallic Zn to the oligomeric deposit. For the sake of comparison, and to optimize the experimental conditions of the asymmetric synthesis, a pristine wire and two modified Zn wires were used for the initial reduction experiments.

The first tested asymmetric reduction of a prochiral molecule, chosen as a model, was the conversion of AP into one of the two enantiomers of 1-phenyl ethanol (Figure 2A). All three wire samples were kept under constant stirring (60 rpm) for one hour inside the respective solutions containing AP. The products were then extracted with heptane from the reaction mixture and analyzed by enantioselective HPLC on a Chirapak IC CSP column, using a circular dichroism (CD) detector (Figures 2B and2C). When 30 mm of oligomer modified wire and 1 mm of bare Zn are immersed in the reaction medium, AP is successfully reduced to (R)-1-phenyl ethanol with a high yield (90%) and an enantiomeric excess (ee) of 85% (Figure 2B red line). This result is confirmed by analyzing the CD chromatograms recorded at a single wave-length during chromatography (Figure 2C), where the first negative signal is related to the preferential production of the (R)-enantiomer, followed by the less intense positive signal of the (S)-antipode. However, when the same experiment was performed by immersing 20 mm of the wire covered with the (R)-oligomer together with 10 mm of bare Zn in the reaction medium, the stereoselectivity of AP reduction drastically decreases (ee < 1 %) (Figure 2B and 2C blue line). This is due to the large amount of bare Zn immersed in the solution, where AP can be spontaneously reduced on the metal surface in a non-enantiospecific way, leading to a racemic product. As a consequence, the two competing reductions, selective and non-selective, take place simultaneously, but the latter with a higher speed at the bare metal, producing both PE enantiomers in almost equal quantities (product yield of (R)-1-PE 41 %, product yield of (S)-1-PE 42 %). This was confirmed by a control experiment (Figure 2B and 2C grey lines) where only a 30 mm long piece of a bare zinc wire was dipped in the precursor solution. In this case, AP was converted as expected into the two PE enantiomers without any selectivity, leading to a racemic mixture. Therefore, the best way to maximize the product yield during the stereoselective synthesis is to expose only a minimum of bare Zn to the reaction medium.

In the light of these findings, two modified Zn wires were prepared by electrodepositing the two antipodes of the BT2T4 oligomer, and immersing them in a 0.5 M sulfuric acid, 100 mM AP solution, with only 1mm of bare Zn and 30mm of modified wire in contact with the reaction medium. The results are depicted in Figures 3A andB. In this way, it was possible to produce preferentially, with a large yield (90%), one or the other PE enantiomer. Perfectly specular chromatograms, with CD detection, were obtained in both cases with an ee value of 85%. This excellent selectivity of the stereospecific synthesis might be explained by the presence of a thin gas layer constantly decorating the fraction of bare Zn, thus preventing the unspecific conversion of the prochiral precursor at this location. Consequently, reduction of the starting compound occurs almost exclusively on the part of the metal surface which is modified with the inherently chiral oligomer.

Another parameter, that could influence the efficiency and selectivity of the process, is the thickness of the deposited chiral oligomer, thus different substrates were prepared by modulating the duration of the galvanostatic electropolymerization. The product mixtures of the asymmetric reduction of AP were analyzed by enantioselective HPLC after one hour, and a decrease in selectivity was observed for thinner deposits (Figure S2). This indicates that the reduction of the prochiral molecule takes place within the polymer matrix and benefits from the chiral environment, which leads to an increase of the ee. In order to verify the general validity of the proposed concept of autonomous enantioselective redox catalysis, the reduction of phenylglyoxylic acid (PGA) to mandelic acid was chosen as a second model reaction (Figure 2A). The same experimental set-up was used for this synthesis. 30 mm of the wire covered by oligo-(R)-BT2T4 were immersed, together with 1 mm of bare Zn, in a 100 mM PGA, 0.5 M H2SO4 solution. As can be seen from the HPLC analysis (Figure 3C and D), (R)-mandelic acid was produced in high yield (88%) with an ee of 79 % demonstrating again the stereospecificity of the process, even in the presence of other functional groups.

Combining stereoselectivity and autonomous motion

As stated above, the spontaneous oxidation of Zn is the main driving force for the reduction of the prochiral molecules on the surface of the inherently chiral monomer. Furthermore, the concomitant bubble formation/release should enable self-propulsion, if the size of the wire is scaled down to smaller dimensions. Therefore, the same strategy of autonomous enantioselective synthesis was followed to design bubble-propelled asymmetric microswimmers. They should not only be able to convert with high selectivity a prochiral compound into its corresponding enantiomer, but also benefit from an efficient renewal of solution at the interface of the swimmer, either by the continuous bubble recoil or by the motion of the device (self-mixing), leading in fine to an increase in production efficiency. The general design of such microswimmers, with the reactions involved in the stereoselective synthesis, is represented in Scheme 2. After the electrochemical deposition of one BT2T4 enantiomer, the functionalized Zn wire was cut into small pieces with an average length of 700 µm ± 400 µm. Scaling down the length of the swimmers, but keeping the diameter identical, might lead to a decrease of %ee, because the amount of wire surface covered by the oligomer becomes comparable with the surface of naked zinc present at the section of the swimmer. However, it is possible to avoid this limitation if not only the length of the swimmers, but also their diameter is decreased simultaneously in order to keep the ratio of polymer-modified and naked surface area constant. Therefore, swimmers with smaller dimensions can in principle be designed by following the same strategy.

From a single wire, two types of swimmers can be produced: Janus type swimmers, with only one bare Zn section, and swimmers with both ends exposing bare Zn (Scheme S1). Since the latter causes an increase in the fraction of metal in direct contact with the solution, one extremity can be isolated with a thin layer of varnish. Consequently, reactions involving the oxidation of the Zn surface will occur only on one side of the swimmer, thus breaking the symmetry of the object and generating motion. Scheme 2. General scheme of a bubble-propelled microswimmer, placed at the air/water interface, together with the redox reactions involved in the enantiospecific conversion.

The autonomous motion and stereoselective synthesis achieved with these Janus-type microswimmers were evaluated by placing carefully 15 of these mobile microreactors at the air/water interface of an aqueous solution of 0.5 M H2SO4 and 100 mM AP (Video S1). Under these conditions, the reduction of protons by Zn dissolution generates the bubbles responsible for the random motion, whereas the enantioselective reduction of prochiral AP to 1-PE is catalyzed by the surface confined enantiopure oligomers. The observed, mostly erratic, motion is caused by the gradual consumption of Zn, breaking the symmetry of hydrogen production on the metal surface.

After one hour of self-mixing, the products were extracted with heptane and analyzed by enantioselective HPLC. The same experiment was also carried out with objects functionalized with the enantiopure oligomer having the opposite configuration. Perfectly specular chromatograms were obtained with HPLC (Figure 4A). The enantioselective synthesis occurred with high yields (92%) and with an ee of 90% in both cases. The red line represents the HPLC result for the enantiospecific process catalyzed by the (R)-oligomer, leading to (R)-1-PE, and the green line stands for the products obtained with the opposite configuration.

Electron Efficiency

Obviously, the reduction of the prochiral starting compound is competing with the production of H2 and both are consuming zinc. However, by definition at least a certain fraction of electrons has to be devoted to gas bubble formation, otherwise there would be no propulsion. Therefore, the overall efficiency of the reaction strongly depends on the ratio between the concentration of reactant and protons, which needs to be optimized. The formation of the desired product via enantioselective reduction and the bubble propulsion mechanism require a constant supply of protons. Thus, at high pH values (pH ≥ 7), the ketone reduction and bubble formation are strongly limited due to the intrinsically low availability of protons in the reaction media. Another drawback of high pH values is related to possible side reactions. Electroreduction of ketones commonly can lead to the production of both, alcohol and pinacol. Even though there are still some open questions concerning the fundamental mechanisms [60] it seems that the reduction of phenyl ketones proceeds via the formation of a free radical as a transition state. [61] At this stage, dimerization of the phenolic radical species might occur, eventually producing by-products. However, the pH of the electrolyte plays a crucial role after this first electron transfer step. [62] If the reaction is carried out in the presence of a sufficiently high concentration of protons, the reaction is shifted to the preferential formation of chiral products during the acceptance of a supplementary electron and proton. We found out that for higher pH values (pH=4), not only the %ee values (%ee=70%), but also the overall yield (73%) decrease, due to the formation of a significant amount of side product. However, when using the optimized pH value of the present work (pH=0), parasitic reactions can be kept to a minimum. Therefore, lower pH values are globally more favorable. For the optimized pH=0, we have also quantified more precisely the fraction of electrons that are really used for the electrosynthesis. Experiments with the static wire without stirring, and also with the 15 microswimmers, were repeated, and the weight of the zinc wire, and of the microswimmers, were measured before and after the synthesis experiments. This allows estimating the amount of zinc that has been consumed and comparing it with the amount of produced chiral molecules. Calculating the ratio between the moles of alcohol produced after 1 h of reaction and the moles of zinc consumed, we found that the percentage of electrons, specifically used for the enantioselective reduction, is 56 % for the static wire, and 75 % for the microswimmers. This means that in both cases a majority of the electrons is directed towards the organic synthesis (Figure S3). These results demonstrate that self-mixing causes a significant increase by 50% of the percentage of electrons employed for the enantioselective reduction, due to an improved mass transport of the prochiral precursor towards the catalytic interface.

Stereoselective Process Efficiency

The efficiency of the stereoselective process can be compared for three different cases: the chiral wire i) in the absence and ii) in the presence of mechanical stirring (60 rpm), and iii) the 15 autonomous swimmers with self-mixing capability. Considering that the combined length of 15 swimmers is approximatively equal to 10.5 mm and that the length of the previously used chiral wires is 31 mm, the conversion rate of AP, normalized by the active length can be calculated for each experiment (see supporting information for details). This results in 0.11 mmol h-1 mm-1 and 0.25 mmol h-1 mm-1 for the chiral wire in the absence and in the presence of mechanical stirring, respectively. Most importantly, the autonomous microswimmers produce the final enantiomer with a rate of 0.7 mmol h-1 mm-1. These values clearly demonstrate a significant improvement in the conversion rate due to the mixing of the solution, inducing a constant supply of educts to the wire.

Since the efficiency is higher with the swimmers, they can be considered as the equivalent of moving microreactors, which are particularly well-adapted to produce the desired enantiomer. The spontaneous random motion leads to active micro-mixing (Figure 4B, Video S1 and S2), thus accelerating the rate of the asymmetric synthesis, and resulting in high yields. This is especially true because the convection occurs all along the active surface of the oligomer with the same intensity, in contrast to the static chiral wire where the mechanical stirring is not efficient enough to improve mass transport everywhere on the catalyst surface, but mostly only at the bottom of the cell.

Using the same experimental conditions for the enantioselective synthesis, the reaction time was further optimized by analyzing the product mixtures obtained after letting move 15 swimmers, functionalized with (R)-oligomer, for different times, ranging from 20 min to 5 h. In Figure S4 the conversion of AP into (R)-and (S)-phenyl ethanol for the first 1.5 hours is represented. AP is specifically transformed into the favored (R)-product, reaching a conversion of 90% after 1 hour. (S)product is also formed in low amounts, with a conversion of 5%. The highest %ee values were obtained after a reaction time of 1 hour (Figure S5). Beyond this time, a decrease in the stereospecificity of the reaction was observed. This is due to the fact that Zn is gradually consumed and the oligomer starts degrading during the synthesis. SEM micrographs of a swimmer, collected from the solution after 2.5 hours show the formation of holes and cracks in the oligomeric substrate (Figure S6). Thus, the solution can infiltrate the oligomer matrix, via the porosity of the film, and reaches the underlying bare Zn surface, causing a more and more pronounced formation of racemic product. This also explains the formation of H2 bubbles not only at the bare zinc section, but also directly on the oligomer surface (Inset Figure 4B). This observation motivated us to evaluate the reusability of the swimmers by filtrating the product mixture to recover all micro-swimmers. A fresh AP solution (100 mM) was prepared, and then the used swimmers were placed again at the air/water interface for another hour. The product mixture was then analyzed by HPLC to evaluate the enantioselectivity of the process. As expected, a decrease in enantioselectivity was observed, but the discrimination between both enantiomers was still very significant (Figure S7), and the selectivity of the swimmers as a function of the number of recycling loops might be further increased by optimizing the thickness of the oligomer layer.

Conclusion

In this work we have designed asymmetric microswimmers by coupling short fractions of a Zn wire, which spontaneously reduces protons, with inherently chiral oligomers for the enantiospecific electroreduction of acetophenone, chosen as prochiral probe molecule. Motion is triggered by bubble propulsion, accompanied by the transport of electrons from Zn to the inherently chiral oligomer surface. The stereoselective synthesis was first optimized by functionalizing macroscopic Zn wires with the enantiopure oligomers. The fraction of bare Zn immersed in solution, and the thickness of the oligomer deposit are crucial parameters to achieve both, high selectivity and production rate. By changing the conformation of the deposited oligomer it is possible to invert in a specular way the selectivity of the asymmetric process. The concept has also been shown to be of general validity by testing another prochiral starting compound, phenylglyoxylic acid.

The same strategy was then explored to design self-propelled chiral swimmers, acting as moving microreactors, able to convert in an enantioselective way the prochiral educt in solution. An ensemble of micro-swimmers, functionalized with one enantiomer of the inherently chiral oligomer, were placed in a solution containing acetophenone and sulfuric acid. An optimization of the reaction time, to obtain the highest selectivity, was performed by analyzing the conversion of acetophenone into the favorite (R)-or (S)-alcohol. The highest values were obtained after 1 hour with a product yield of 92 % and an %ee of 90% for both swimmers functionalized with chiral oligomers of opposite conformation. Moreover, and most importantly, the normalized conversion rate of acetophenone, which can be achieved with the microswimmers, is significantly higher compared to what can be obtained with the static wire, and also higher even when the static wire is employed together with mechanical stirring. This clearly indicates the added value of using such mobile micro-reactors in electroorganic synthesis, as they continuously and autonomously stir their local environment, thus ensuring an efficient mass transport of educt towards the swimmer surface.

In conclusion, these original systems appear particularly efficient for asymmetric reductions in order to obtain, with high specificity, the desired enantiopure product with excellent yield. Their performance is enhanced by the fact that the autonomous motion, based on a bubble-propulsion mechanism, induces active micro-mixing, which leads to a more efficient renewal of the prochiral starting compound at the entire catalyst interface. Such an active mixing helps overcoming one of the potential drawbacks of conventional electroorganic synthesis. [5][6][7] Moreover, since the oligomeric antipodes that constitute the catalytic layer are available in both conformations, it is possible to generate selectively both enantiopure products. Finally, with respect to future applications, these catalytically active autonomous chiral swimmers could be potentially used enmasse to scale-up asymmetric reduction processes. However, taking into account that the coated Zn bodies will be consumed by the reaction, it will be necessary to optimize the reaction time, and to fine-tune the ratio between reducing agent and prochiral starting compound.

Figure 1 .

 1 Figure 1. EDS characterization of two selected sections of a Zn wire functionalized with oligomer (orange) and bare Zn (grey). Inset: SEM micrograph of the junction between bare Zn and oligomer modified Zn, employed for the enantioselective catalysis.

Figure 2 .

 2 Figure 2. (A) Asymmetric reductions chosen as model reactions. (B) and (C) Chromatograms with online UV/CD detection recorded after the conversion of acetophenone (100 mM) into (R)-or (S)-1phenylethanol by soaking in a 0.5 M H2SO4 solution: i) 30 mm of the (R)-BT2T4 oligomer modified wire and 1 mm of bare zinc (red line); ii) 20 mm of the (R)-oligomer modified wire and 10 mm of bare zinc (blue line) and iii) 30 mm of a pristine zinc wire (grey line).

Figure 3 .

 3 Figure 3. (A) and (B) Chromatograms with online UV/CD detection recorded after the same synthesis as in Figure 2 with 1 mm of bare zinc in contact with the solution, but the wires were functionalized with (R)-BT2T4 oligomer (red lines) and with (S)-BT2T4 oligomer (green lines). (C) and (D) Chromatograms with online UV/CD detection recorded after the conversion of 100 mM phenylglyoxylic acid into mandelic acid. The wire was functionalized with (R)-BT2T4 oligomer and soaked in 0.5 M H2SO4 solution.

Figure 4 .

 4 Figure 4. (A) Chromatograms recorded with the product mixtures obtained from the asymmetric synthesis carried out by placing 15 microswimmers, functionalized with (R)-BT2T4 oligomer (red line) and (S)-BT2T4 oligomer (green line), in a 100 mM acetophenone 0.5 M H2SO4 solution. (B) Trajectory plot of a microswimmer functionalized with the (R)-enantiopure oligomer placed in the same solution as in (A), tracked during a time interval of 15 min. The inset shows a close-up of a swimmer indicating bubble formation at the bare Zn surface (left extremity), but also to a smaller extent on the oligomer modified section.

Acknowledgements

The work has been funded by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement n° 741251, ERC Advanced grant ELECTRA). S.A. acknowledges financial support of Università degli Studi di Milano. The authors are also very grateful for fruitful discussions with Patrizia Mussini.


Guillermo Angeles
email: guillermo.angeles@inecol.mx

Marycruz Álvarez-Jiménez

Fabien Anthelme

Isabelle Barois

José Luis

Chaparro Carrillo

Nereyda Cruz-Maldonado

Thibaud Decaëns

Martin De Los Santos-Bailon

Stéphane Fourtier

Emmanuel Garcia-Gutierrez

Thibaud Decaens

Martín De Los Santos-Bailón

Emmanuel García-Gutiérrez

David García-Segura

Rodrigo Gómez-Aguilar

Antonio Gómez-Anaya

Eduardo Hernández

Daniel Hernández-Cáceres

Leonor Jiménez

Beatriz E Marín-Castro

Luis Merino-Martín

Marco A Morales-Martínez

Edwin U Monfil-León

René A Palestina

Carlos Pimentel-Reyes

Ricardo Quiroz Reyes

Frédérique Reverchon

Hervé Rey

Catherine Roumet

Christina D Siebe-Grabach

Katrin Sieron

Víctor Vázques

Monique Weemstra

Alexia Stokes

Drivers of soil biophysical processes along an elevational gradient at Pico de Orizaba volcano, Mexico

Keywords: Elevational gradient, Bulk soil and rhizosphere microbial communities, Carbon storage, High mountain ecosystem, Soil biodiversity, Soil hydrophysical properties, Soil macro and meso-fauna, Soil structure and soil moisture monitoring

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

Background

Elevational gradients and the associated biotic and abiotic changes provide useful frameworks to elucidate the drivers behind species distribution and their response to climate change [START_REF] Cruz-Maldonado | Aboveground-trait variations in 11 (sub)alpine plants along a 1000-m elevation gradient in tropical Mexico[END_REF][START_REF] Fukami | Long-term ecological dynamics: reciprocal insights from natural and anthropogenic gradients[END_REF][START_REF] Mccain | Elevational gradients in species richness[END_REF]. Environmental factors such as temperature, radiation, and precipitation shift with altitude and strongly influence soil biophysical properties, including soil structure and moisture. Plant communities also change along altitudinal gradients, further modifying soil properties through litter and root carbon (C) inputs, thereby affecting soil biodiversity. Here, we collected belowground and aboveground data to elucidate the mechanisms through which altitude and the associated shifts in climate and vegetation affect soil properties and ecosystem processes, at different elevations.

The ecosystem services provided by (sub)alpine soils, e.g., C sequestration, nutrient cycling, and water retention, are threatened by a changing climate and land use conversion, as high mountains are particularly fragile ecosystems (Mountain Research Initiative 2015). These ecosystem services depend on soil physicochemical properties, its biodiversity, as well as on plant species composition and community structure. Therefore, our dataset includes data on soil porosity and aggregate stability at different soil depths and altitudinal levels and measurements of soil water infiltration and retention, which will allow a better understanding of the water flow dynamics along this Neotropical altitudinal gradient. We also collected data on soil macro-and meso-fauna diversity, as well as on microbial communities at the bulk soil and rhizosphere level, thus allowing researchers to explore the biotic and abiotic drivers behind soil biodiversity, which is key in mediating soil biophysical processes [START_REF] Collins | Belowground impacts of alpine woody encroachment are determined by plant traits, local climate and soil conditions[END_REF][START_REF] Hernández-Cáceres | Vegetation creates microenvironments that influence soil microbial activity and functional diversity along an elevation gradient[END_REF]. This dataset complements the already published data from our twin study in France [START_REF] Stokes | Shifts in soil and plant functional diversity along aaltitudinal gradient in the French Alps[END_REF]) and represents the largest freely available collection of data on plant traits, soil physicochemical variables and soil biodiversity along a 1200 m elevational gradient in a Neotropical climate. Methods for data collection and analysis are provided in the data files and supplementary materials (Table 1).

Objectives

Our objective was to determine how changes in environmental conditions with altitude affect edaphic properties, vegetation, and soil microbial and faunal communities, by collecting data along a 1200 m long gradient between 3400 and 4600 m.a.s.l. on the North-East flank of the highest mountain of Mexico, Pico de Orizaba volcano.

A twin study with the same objective was carried out at Massif de Belledonne, in the French Alps.

Methods

Data file 1 shows an aerial map of the whole protected area named "Área Natural Protegida Pico de Orizaba (ANPPO)", within which our study sites are located. Data file 1 also provides the names and numbers of each plot, with their respective coordinates and elevations.

The studied elevational gradient lies on the North-East flank of Pico de Orizaba, the highest peak of Mexico. We hereby present data collected in 2017 and 2018, at six altitudes (elevational bands of 200 m each) along an elevational gradient ranging from 3400 to 4600 m.a.s.l. and from 3400 to 4400 m.a.s.l. for soil profile and soil moisture (Data files 2 and 4, respectively), at Pico de Orizaba volcano, Mexico. Climatic data (Data file 3) are also provided and were measured over 2 years (2018)(2019)(2020).

The underlying bedrock is composed of intermediate to acid lavas and pyroclastic (volcaniclastic) material. Soil profiles were described for each altitudinal level (two soil profiles made per altitudinal band). Soil physico-chemical properties were analyzed from each identified soil horizon. The main soil units correspond to Endosilandic Andosols at Sleketic Regosol. The volumetric and tension soil moisture, as well as the soil surface temperature were monitored for one year, in six soil profiles distributed along the altitudinal gradient (Data file 4).

To obtain the floristic list of our study site, five 20 × 20 m plots were established at each altitude (every 200 m along our 1000 m long elevational gradient) up to 4000 m of elevation, to include keystone and communitystructuring species with different growth forms: Pinus hartwegii (tree), Oxylobus arbutifolius and Chionolaena lavandulifolia (shrubs), and Lupinus montanus and Senecio roseus (herbs). From 4200 m.a.s.l. and above, the distance between our plots had to be reduced to 5 m for space availability reasons (mountain tapering). A botanical survey was performed inside each plot (Data file 5) and one adult individual of each structuring species was selected per plot. At the limit of the individual's canopy on the downslope side, infiltration tests were performed to estimate water flow through soil and hydraulic conductivity of the quasi-steady phase was calculated (Data file 6). Aggregate stability measurements were made from soil blocks (10 × 10 × 15 cm depth) taken beside the monoliths (Data file 7). An earthworm survey was made on soil monoliths of 0.25 × 0. 25 × 0.15 m in the field to link the abundance of these ecosystem engineers with soil processes (Data file 8). To investigate the relationships between soil biophysical properties and vegetation, plant community composition was described in a 1 m 2 subplot within each plot, which was established near each structuring species (Data file 10). Soil texture, bulk and real density, cationic exchange capacity, pH, electric conductivity, organic C, nitrogen (N) content, nitrate and ammonium concentrations, total and available phosphorus (P), and macronutrients (Ca, Mg, K, Na) concentrations were determined on pooled soil samples harvested within each monolith (Data file 9). A soil monolith (0.25 m × 0.25 m × 0.15 m depth) was excavated within each subplot (n = 83). Above the monolith, litter layer thickness and aboveground biomass per species were measured (Data file 10). Soil samples were collected from the monolith and from rhizospheric soil attached to fine roots of the three structuring plant species. DNA was extracted from these soil samples and 16S rDNA amplicon sequences were obtained to determine the changes in bacterial communities at the bulk soil and at the rhizosphere level along the elevational gradient (Data file 11). Soil mesofauna was collected beside each monolith from litter and soil with a cylinder (5 cm diameter and 10 cm high) and then extracted with a Berlese funnel in the laboratory. All the organisms were kept in 70% alcohol and identified at order taxonomical level, except for Collembola for which identification was made at the family level (Data file 12). Soil macroinvertebrates were hand-sorted from each monolith and fixed in 70% ethanol, except earthworms, which were fixed in 96% ethanol for the DNA barcoding. Macroinvertebrates were identified at the order level, except for earthworms for which morphological diagnoses were combined with DNA barcoding to obtain species level assignations. Invertebrates within each taxon were counted and weighed (Data file 13).

Limitations

Although this dataset comprises a large number of field and laboratory data collected from 83 monoliths in 30 plots, it was not always possible to include all structuring species within the same plot. At higher altitudinal levels (4000-4400 m.a.s.l.), additional shrub and herbaceous species were sometimes included (S. roseus and C. lavandulifolia) to complement the data when L. montanus and O. arbutifolius were not present. Moreover, our focus on some keystone/structuring plant species does not allow generalization of rhizosphere processes under other species. Finally, our datasets do not provide information on plant functional traits nor on soil microbial functional diversity (i.e., enzymatic activities or microbial gene transcripts), the latter being a key factor to understand soil functioning as microorganisms are main contributors to soil biophysical processes.

Conclusion

Our data allow researchers to elucidate the changes in soil ecosystem services and their link with soil biodiversity along an altitudinal gradient in the Neotropical region, where high mountain ecosystems have been poorly studied. Under a climate change context, these data are particularly useful to improve our predictions of possible shifts in vegetation and soil organisms.

Table 1

 1 Overview of data files/data sets. Pico de Oizaba, Mexico

Label Name of data file/dta set File type (file extension) Data repository and identifier (DOI)

	Data file 1	Plots and map	MS Excel file (.xlsx)	10.6084/
				m9.figshare.22057106 https:// figsh
				are. com/s/ d474d a60e1 5dbb9 37251
	Data file 2	Soil profile	MS Excel file (.xlsx)	10.6084/
				m9.figshare.22097993 https:// figsh
				are. com/s/ 4e917 b0316 48148 a7f87
	Data file 3	Plot climate interpolated base	MS Excel file (.xlsx)	10.6084/
				m9.figshare.22057175 https:// figsh
				are. com/s/ 369a8 0465b 62cfa 15249
	Data file 4	Soil moisture monitoring station	MS Excel file (.xlsx)	10.6084/m9.figshare.22102379 https://
				figsh are. com/s/ e9c60 5c45c ea45b
				3a669
	Data file 5	Floristic list	MS Excel file (.xlsx)	10.6084/m9.figshare.22010852 https://
				figsh are. com/s/ f6407 9ca0a 9bf3c
				eebe7
	Data file 6	Infiltration and field saturated hydraulic conductivity	MS Excel file (.xlsx)	10.6084/
				m9.figshare.22093580 https:// figsh
				are. com/s/ f6407 9ca0a 9bf3c eebe7
	Data file 7	Monolith aggregate stability	MS Excel file (.xlsx)	10.6084/
				m9.figshare.22010849 https:// figsh
				are. com/s/ ce7b2 dcf96 a58b8 48fdf
	Data file 8	Monolith earthworms	MS Excel file (.xlsx)	10.6084/
				m9.figshare.22057160 https:// figsh
				are. com/s/ 629b3 fcaaf cd994 bb5e5
	Data file 9	Monolith soil analysis	MS Excel file (.xlsx)	10.6084/
				m9.figshare.22010846 https:// figsh
				are. com/s/ dd175 f66a0 54808 a8c2b
	Data file 10	Monolith vegetation	MS Excel file (.xlsx)	10.6084/m9.figshare.22105787 https://
				figsh are. com/s/ b1565 9ca00 7b508
				31440
	Data file 11	Bulk soil and rhizosphere 16S amplicon sequences	MS Excel file (.xlsx)	10.6084/
				m9.figshare.22062104 https:// figsh
				are. com/s/ 4b0a2 ae602 90939 0c1b7
	Data file 12	Monolith macrofauna	MS Excel file (.xlsx)	10.6084/m9.figshare.22065185 https://
				figsh are. com/s/ ea9cb f6b1d 8708e
				a8674
	Data file 13	Soil mesofauna	MS Excel file (.xlsx)	10.6084/
				m9.figshare.22065302 https:// figsh
				are. com/s/ c9f29 1f23d 0cf4b 8cc2d

Funding

Funding was provided by the French and Mexican governments (ECOPICS project, ANR-16-CE03-0009 and CONACYT-2 73659).

Availability of data and materials

The data described in this Data note can be freely and openly accessed on https:// figsh are. com/. Please see Table 1 for details and links to the data.

Abbreviation m

Meters above sea level

Author contributions

All authors contributed to data collection and writing of the paper. All authors read and approved the final manuscript.

Declarations

Ethics approval and consent to participate Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Weiwei Li

Chunhua Zhang

Ziman Zheng

Xiaoyu Zhang

Lin Zhang

Alexander Kuhn

Fine-tuning the Electrocatalytic Regeneration of NADH Cofactor using [Rh(Cp*)(bpy)Cl] + Functionalized Metal-Organic Framework Films

Keywords: NU-1000 film, electroenzymatic synthesis, electrocatalysis, redox hopping, NADH regeneration

Electrochemical regeneration of the reduced form of the nicotinamide adenine dinucleotide (NADH) cofactor catalyzed by immobilized [Rh(Cp*)(bpy)Cl] + is a promising approach for enzymatic synthesis of many valuable chemicals with NAD-dependent dehydrogenases.

However, a rational control of the efficiency is often limited by the irregular structure of the electrode/electrolyte interface and the accessibility of the molecular catalyst. Here, we propose an electrochemical system for NADH cofactor regeneration, based on highly ordered 3D metalorganic framework (NU-1000) films. [Rh(Cp*)(bpy)Cl] + is incorporated at the zirconium nodes of NU-1000 via solvent-assisted ligand incorporation (SALI), leading to a diffusion-controlled behavior, associated with an electron hopping mechanism. Varying the ratio of redox-active [Rh(Cp*)(bpy)Cl] + and inactive post-grafting agents enables the elaboration of functional electrodes with tunable electrocatalytic activity for NADH regeneration. The exceptionally high Faradaic efficiency of 97%, associated with a very high turnover frequency (TOF) of ~1400 h -1 for NADH regeneration, and the total turnover number (TTN) of over 20000 for the enzymatic conversion from pyruvate to L-lactate, when coupled with L-Lactate dehydrogenases (LDH) as a model reaction, open up promising perspectives for employing these electrodes in various alternative bioelectrosynthesis approaches.

INTRODUCTION

Oxidoreductases have been widely used for bio-transformations due to their high activity and enantioselectivity under mild operating conditions. The regeneration of the reduced form of the nicotinamide adenine dinucleotide (NADH) cofactor is of great interest for potential industrial applications of enzymatic synthesis, due to the fact that around 80% of known oxidoreductases are nicotinamide dinucleotide (NAD) dependent enzymes. However, the expensive NADH cofactor is a crucial ingredient, since it directly participates in the enzymatic redox reactions. [START_REF] Simon | Biomolecular Films: Design, Function,and Applications[END_REF] Thus, over the last decades, the regeneration of NADH has been intensively investigated by enzymatic, chemical, photochemical, [START_REF] Liu | Single Cobalt Atom Anchored on Carbon Nitride with Well-Defined Active Sites for Photo-Enzyme Catalysis[END_REF][START_REF] Zhang | Bioinspired Photocatalytic NADH Regeneration by Covalently Metalated Carbon Nitride for Enhanced CO 2 Reduction[END_REF] and electrochemical methods. [START_REF] Lee | Advances in Electrochemical Cofactor Regeneration: Enzymatic and Non-Enzymatic Approaches[END_REF] The electrochemical regeneration of NADH can be considered as an attractive concept, since electricity can originate from sustainable resources and therefore enables in principle a green chemistry approach with low cost. However, the direct electrochemical reduction of NAD needs rather high overpotentials, leading to the formation of biologically inactive NAD 2 dimers or isomers instead of the active 1,4-NADH in a two-step reaction mechanism. [START_REF] Gorton | Electrochemistry of NAD(P) + /NAD(P)H. In Encyclopedia of Electrochemistry[END_REF] In order to obtain 1,4-NADH, different electrocatalytic systems were proposed and established. [START_REF] Lee | Advances in Electrochemical Cofactor Regeneration: Enzymatic and Non-Enzymatic Approaches[END_REF] Among them, the organometallic catalyst [Rh(Cp*)(bpy)Cl] + belongs to the most efficient ones and has been immobilized on electrodes, not only to increase the reusability of the catalyst, but also to prevent undesirable interactions between the functional groups of enzymes and [Rh(Cp*)(bpy)Cl] + . [START_REF] Walcarius | Factors Affecting the Electrochemical Regeneration of NADH by (2,2'-Bipyridyl) (Pentamethylcyclopentadienyl)-Rhodium Complexes: Impact on Their Immobilization onto Electrode Surfaces[END_REF] The rhodium functionalized electrodes have been prepared, among others, by depositing polymers bearing Rh moieties, [START_REF] Hildebrand | Stable Electroenzymatic Processes by Catalyst Separation[END_REF] or using - stacking interactions with carbon materials, [START_REF] Tan | Regeneration of the NADH Cofactor by a Rhodium Complex Immobilized on Multi-Walled Carbon Nanotubes[END_REF] as well as through the covalent immobilization of Rh catalysts on the electrode surfaces. [START_REF] Zhang | Electrocatalytic Biosynthesis Using a Bucky Paper Functionalized by [Cp*Rh(Bpy)Cl] + and a Renewable Enzymatic Layer[END_REF][START_REF] Zhang | Bulk Electrocatalytic NADH Cofactor Regeneration with Bipolar Electrochemistry[END_REF][START_REF] Zhang | Covalent Immobilization of (2,2′-Bipyridyl) (Pentamethylcyclopentadienyl)-Rhodium Complex on a Porous Carbon Electrode for Efficient Electrocatalytic NADH Regeneration[END_REF][START_REF] Zhang | Molecular and Biological Catalysts Coimmobilization on Electrode by Combining Diazonium Electrografting and Sequential Click Chemistry[END_REF] Efficient NADH regeneration rates were obtained, but due to the irregular porosity of the final electrode layer (CNTs, polymers) and an uncontrolled position of the molecular catalyst units after immobilization via adsorption, encapsulation or diazonium grafting, it is difficult to predict and fine-tune the efficiency of the electrode. Thus, a rational design of the porous structure and a precise control of catalyst loading is highly desirable for the development of efficient functional bioelectrodes. Different types of metal-organic framework materials with porous crystalline structures were explored for a variety of potential applications [START_REF] Lin | Bioinspired Metalation of the Metal-Organic Framework MIL-125-NH 2 for Photocatalytic NADH Regeneration and Gas-Liquid-Solid Three-Phase Enzymatic CO 2 Reduction[END_REF][START_REF] Xia | HKUST-1 Catalyzed Efficient in Situ Regeneration of NAD + for Dehydrogenase Mediated Oxidation[END_REF] , including in the field of electrochemistry. [START_REF] Al-Kutubi | Electrosynthesis of Metal-Organic Frameworks: Challenges and Opportunities[END_REF][START_REF] Yadnum | Site-Selective Synthesis of Janus-Type Metal-Organic Framework Composites[END_REF][START_REF] Suttipat | Chiral Macroporous MOF Surfaces for Electroassisted Enantioselective Adsorption and Separation[END_REF][START_REF] Warakulwit | Elaboration of Metal Organic Framework Hybrid Materials with Hierarchical Porosity by Electrochemical Deposition-Dissolution[END_REF][START_REF] Xie | Electrically Conductive Metal-Organic Frameworks[END_REF] NU-1000 is a zirconium-based metal-organic framework with a modular porous structure, which can be tailored by different functional groups. [START_REF] Wang | Scalable Synthesis and Post-Modification of a Mesoporous Metal-Organic Framework Called NU-1000[END_REF] Its high surface area and good stability in the aqueous phase makes it a good candidate as a support for catalyst immobilization in biosystems. NU-1000 or its derivatives have been used for the encapsulation of enzymes such as cutinases, [START_REF] De Ruiter | Hierarchical Pore Structures as Highways for Enzymes and Substrates[END_REF] cytochrome c, [START_REF] Chen | Insights into the Enhanced Catalytic Activity of Cytochrome c When Encapsulated in a Metal-Organic Framework[END_REF][START_REF] Sha | Stabilization of an Enzyme Cytochrome c in a Metal-Organic Framework against Denaturing Organic Solvents[END_REF] and formate dehydrogenase, [START_REF] Chen | Stabilization of Formate Dehydrogenase in a Metal-Organic Framework for Bioelectrocatalytic Reduction of CO 2[END_REF] leading to enhanced stability and catalytic response. The immobilization of NU-1000 metal-organic framework films on electrodes has been proposed and studied in the field of electroanalysis, [START_REF] Chuang | Metal-Organic Frameworks toward Electrochemical Sensors: Challenges and Opportunities[END_REF][START_REF] Biswas | Polypyrrole Merged Zirconium-Based Metal-Organic Framework NU-1000 for Detection of Levodopa[END_REF] electrocatalysis [START_REF] Sanati | Enhanced Electrochemical Oxygen and Hydrogen Evolution Reactions Using an NU-1000@NiMn-LDHS Composite Electrode in Alkaline Electrolyte[END_REF][START_REF] Noh | Redox-Mediator-Assisted Electrocatalytic Hydrogen Evolution from Water by a Molybdenum Sulfide-Functionalized Metal-Organic Framework[END_REF] and energy storage. [START_REF] Liu | Improving Charge Transfer in Metal-Organic Frameworks through Open Site Functionalization and Porosity Selection for Li-S Batteries[END_REF] Since the initial NU-1000 is an insulator, the electron transfer through NU-1000 films can be in general improved by following two approaches. The first option is to incorporate a conductive material into the pores of MOFs (conductive guests, [START_REF] Talin | Tunable Electrical Conductivity in Metal-Organic Framework Thin-Film Devices[END_REF] C 60 , [START_REF] Goswami | A Porous, Electrically Conductive Hexa-Zirconium(Iv) Metal-Organic Framework[END_REF] conducting polymers, [START_REF] Wang | Rendering High Surface Area, Mesoporous Metal-Organic Frameworks Electronically Conductive[END_REF] etc.). The second elegant way is to functionalize the MOF nodes or linkers with redox active moieties allowing redox hopping of charges. [START_REF] Shimoni | Tuning of Redox Conductivity and Electrocatalytic Activity in Metal-Organic Framework Films Via Control of Defect Site Density[END_REF][START_REF] Lin | The Role of Redox Hopping in Metal-Organic Framework Electrocatalysis[END_REF][START_REF] Ahrenholtz | Solvothermal Preparation of an Electrocatalytic Metalloporphyrin MOF Thin Film and Its Redox Hopping Charge-Transfer Mechanism[END_REF][START_REF] Hod | Modulating the Rate of Charge Transport in a Metal-Organic Framework Thin Film Using Host:Guest Chemistry[END_REF] When comparing the electrical conductivity of an initial NU-1000 film along different axes of the network, three orders of magnitude higher hopping coefficients are measured when the orientation of the MOF crystal is perpendicular to the electrode surface compared to a parallel alignement. [START_REF] Goswami | Anisotropic Redox Conductivity within a Metal-Organic Framework Material[END_REF] Therefore, it is essential to control the orientation of NU-1000 crystals on the conductive substrates. The presence of carboxyl groups on the substrate has been used for controlling the orientation of MOFs. Different substrates (Si, Ti [START_REF] Van Gough | Controlled Nucleation and Growth of Pillared Paddlewheel Framework Nanostacks onto Chemically Modified Surfaces[END_REF] , Au 39,40, and FTO [START_REF] Verma | Controlling Polymorphism and Orientation of NU-901/NU-1000 Metal-Organic Framework Thin Films[END_REF][START_REF] Chuang | Redox-Hopping and Electrochemical Behaviors of Metal-organic Framework Thin Films Fabricated by Various Approaches[END_REF] ) have been modified with carboxyl groups before growing a NU-1000 layer, leading to a preferential perpendicular growth. Among all possible flat electrode materials, glassy carbon (GC) is one of the most suitable for the electrochemical regeneration of NADH, due to its stability and wide potential window, but the poor adhesion of films limits its application as a support. However, molecular surface functionalization can be used as an efficient strategy to improve the adhesion of films on GC. [START_REF] Phal | Electrografting of 4-Carboxybenzenediazonium on Glassy Carbon Electrode: The Effect of Concentration on the Formation of Mono and Multilayers[END_REF] Therefore, the introduction of carboxyl groups on a GC surface will not only help orientating the film growth, but also act as a stabilizing element with respect to the adhesion of a NU-1000 film. In addition, the functionalization strategy can be extended to macroporous carbon for further applications in biosynthesis.

Based on these considerations, we propose here to establish a bioelectrochemical system for the regeneration of NADH, catalyzed by a NU-1000 film grown on glassy carbon (GC) as shown in Scheme 1.

GC was first functionalized with carboxyl groups by electrooxidation of 4-aminobenzoic acid (GC-COOH). Then the formation of a NU-1000 film was triggered by a coordination step with a Zr-oxo cluster (GC-Zr), followed by a solvothermal synthesis step in precursor solution (GC-NU1000). The resulting NU-1000 film serves as a 3D framework with regularly positioned nodes, acting as a catalyst loading points. The carboxyl-functionalized Rh catalyst (Rh-COOH) can then be anchored onto the nodes of the film by solvent-assisted ligand incorporation (SALI).

Taking advantage of the MOF structure, we expect to achieve the rational incorporation of a predefined amount of Rh, leading to a tunable efficiency for cofactor regeneration. The [Rh(Cp*)(bpy)Cl] + loading of the film can be tuned and characterized by changing the initial concentration ratio between the redox-active Rh-COOH and the redox-inactive biphenyl-4carboxylic acid during the SALI process. The electrocatalytic performance of the resulting surface layer towards NADH regeneration can then be characterized in terms of Faradic efficiency and turnover frequency of the total system. Finally, this electrode can be applied for the electroenzymatic conversion from pyruvate to L-lactate, in the presence of L-Lactate dehydrogenases as a model enzyme.

EXPERIMENTAL METHODS

Reagents and materials

Tris (hydroxymethyl) aminomethane hydrochloride ((Tris-HCl), 99%, Macklin); β-Nicotinamide adenine dinucleotide ((β-NAD + ), ≥97%, Aladdin); β-Nicotinamide adenine dinucleotide, reduced disodium salt hydrate ((β-NADH), ≥97%, Aladdin); alcohol dehydrogenase from Saccharomyces cerevisiae (≥300 U/mg, Sigma-Aldrich); L-lactate dehydrogenase (LDH, ≥200 U/mg, Sigma-Aldrich); Sodium pyruvate (98%, Aladdin); Sodium lactate (98%, Aladdin); 1,3,6,8-tetrakis (p-benzoic acid) pyrene ((H 4 TBAPy)(98%, Energy Chemical); biphenyl-4-carboxylic acid (98%, Energy Chemical); zirconium (IV) oxychloride octahydrate (99%, Energy Chemical); ferrocene (98%, Sigma-Aldrich); pentamethylcyclopentadienylrhodium (III) chloride dimer ((RhCp*Cl 2 ) 2 , ≥ 97%, Sigma-Aldrich); 2,2'-bipyridine-4-carboxylic acid (98%, Macklin); acetone (AR, Sinopharm); N,N-dimethylformamide ((DMF), AR, Kermel); tetrabutylammonium hexafluorophosphate ((TBAPF 6 ) 98%, Sigma); 4aminobenzoic acid (99%, Sigma); acetonitrile (AR, Macklin); methanol (AR, Tianjin Fuyu), silver nitrate (AR, Sinopharm reagent); tetrabutylammonium tetrafluoroborate ((TBAFB), 98%, Aladdin,); acetaldehyde (AR, Sinopharm); potassium ferricyanide (99.5%, Tianjin guangfu ); Glassy carbon electrodes were purchased from Tianjin aida.

Apparatus

The morphology and the energy dispersive X-ray spectroscopy (EDS) of the sample was characterized by a Field Emission Scanning Electron Microscope (GeminiSEM-500 Zeiss), and a Scanning Transmission Electron Microscopy (STEM, JEM-F200, JEOL, Akishima, Japan). The X-ray photoelectron spectroscopy (XPS) was performed with a PerkinElmer PHI 5000. The Ultravioletvisible (UVvis) spectra were obtained using a UV-2600 spectrophotometer (Shimadzu, Japan). The powder X-ray diffraction (PXRD) was carried out with a DX-2700BH (Cu Kα, λ = 1.5418 Å). BET adsorption was measured with a high precision gas/vapor adsorption measurement instrument (BELSORP-maxⅡ) and the pore size measurement was carried out by using the instrument's inbuilt nonlocal density functional theory (NLDFT) model.

The ultrathin section was obtained with a Leica EM UC7 Ultramicrotome. High performance liquid chromatography (HPLC) was carried out with an Agilent 1260 Infinit. The column was a Hi-plex H (7.7 × 300 mm, particle size is 8 μm).

All electrochemical experiments were carried out using an Autolab PGSTAT302N. Ag/AgCl (3.5 M KCl) and Ag + /Ag (0.01 M AgNO 3 in 0.1 M TBAFB acetonitrile) were used as reference electrodes in the aqueous and organic phase, respectively. A Pt wire was used as the counter electrode.

Preparation of GC-NU1000

The glassy carbon (GC) electrode was polished with 1000 mesh sandpaper and then washed in deionized water under ultrasonication for 30 s. Then carboxyl (-COOH) groups were generated on the GC electrode by electrochemical oxidation of 1 mM 4aminobenzoic acid in acetonitrile. The electrochemical oxidation was conducted by running 20 cycles of cyclic voltammograms (CVs) ranging from 0 V to 1.6 V at a scan rate of 100 mV s -1 . Then the GC-COOH electrode was washed under ultrasonication with deionized water and ethanol. Finally, the electrode was dried for further use. After dissolving ZrOCl 2 •8H 2 O (0.1 g) in 10 mL DMF the GC-COOH electrode was vertically placed in the solution and heating at 80°C in the oven for 1 h. After taking it out from the solution, the electrode was washed with DMF under ultrasonication. The film growth was carried out following a literature report . [START_REF] Webber | Role of a Modulator in the Synthesis of Phase-Pure NU-1000[END_REF] 0.39 g ZrOCl 2 • 8H 2 O and 17.57 g biphenyl-4carboxylic acid were dissolved in 32 mL DMF in a 100 mL round bottom flask. The solution was incubated at 80°C for 1 h and then cooled down to room temperature. Subsequently, 150 mg of 1, 3, 6, 8-tetrakis (p-benzoic acid) pyrene (H 4 TBAPy) was dissolved in 30 mL of the above solution. The in-situ growth of the NU1000 film on the GC electrode (GC-NU1000) occurred by placing the GC-Zr electrode vertically in the above prepared solution. The bottle was incubated in an oil bath at 100°C for 24 h. Then the modifier biphenyl-4-carboxylic acid was removed by soaking the GC-NU1000 electrode in a solution, prepared by adding 0.5 mL 8 M HCl to 12 mL DMF at 100°C in the oven for 48 h under shaking. Finally, the electrode was soaked in acetone for 12 h in order to remove residual solvent.

Functionalization of GC-NU1000 with Rh catalyst

Carboxyl functionalized [Rh(Cp*)(bpy)Cl] + (Rh-COOH) was immobilized at the nodes of the NU-1000 film via solvent-assisted ligand incorporation (SALI) following a literature report. [START_REF] Deria | Versatile Functionalization of the Nu-1000 Platform by Solvent-Assisted Ligand Incorporation[END_REF] The solutions were prepared by mixing different ratios (0%, 10%, 50%, and 100%) of Rh-COOH with biphenyl-4-carboxylic acid. The total molar concentration of Rh-complex and biphenyl-4-carboxylic acid was kept constant for all experiments at a value of 2 mM, and the mixture was dissolved in 10 ml DMF. The SALI process was induced by dipping the GC-NU1000 electrode in the solution at 60 °C for 24 h under stirring. The obtained GC-NU1000-Rh electrodes were rinsed with acetone.

RESULTS AND DISCUSSIONS

NU-1000 films have been grown on GC via a sequence of several steps. As indicated above, the first step consists in the covalent grafting of a carboxyphenyl group, followed by the complexation with a Zr-oxo unit. The accomplishment is supported by XPS measurements. As shown in Figure 1A, the high-resolution C 1s spectra display characteristics peaks at 288.6, 287.0, 285.4 eV, corresponding to the O-C=O, C=O and C-OH functions of the carboxyl group, [START_REF] Phal | Electrografting of 4-Carboxybenzenediazonium on Glassy Carbon Electrode: The Effect of Concentration on the Formation of Mono and Multilayers[END_REF] and the peak at 284.8 eV can be attributed to the glassy carbon substrate.

The relative increase of the characteristic peak at 288.6 eV, compared to bare GC, indicates the successful attachment of the carboxyphenyl groups to the surface of the GC-COOH electrode.

The Zr 3d spectra of the GC-Zr electrode exhibits two peaks at 185.5 eV and 183.1 eV, which can be assigned to Zr 3d 3/2 and Zr 3d 5/2 , suggesting that the Zr-oxo clusters are immobilized through a reaction with the carboxyl groups. The electrochemical characteristics of the electrodes after carboxyphenyl modification were also tested by performing cyclic voltammetry with two kinds of electrochemical probes (Figure S1). The redox peaks of the negatively charged probe Fe(CN) 6 3-almost completely disappear after carboxyphenyl functionalization due to electrostatic repulsion, while the redox peaks are identical before and after grafting of the molecular layer when ferrocene is used as a neutral probe. This indicates that the functional layer is present on the GC electrode, but also, and most importantly, that the electron transfer is not inhibited by its presence. The surfaces, pre-conditioned in this way, were then employed to grow crystalline rods of NU-1000, resulting in a film with a uniform coverage on the electrode. Figure 1B electrode is shown in Figure S2A, illustrating again the overall homogeneity of the sample. In an important control experiment, a NU-1000 film has been grown on a bare GC electrode without carboxyphenyl functionalization. Figure S2B shows that in this case the NU-1000 film does not grow in a dense and uniform way. Unfortunately it is not possible to gradually tune the density and orientation of the MOF growth on the electrode by changing the density of the carboxyphenyl layer. Either there is a full layer or no layer at all, so fine-tuning is not possible based on this parameter. However, fine-tuning can be achieved by varying the degree of Rh loading of the MOF for a well-oriented GC-NU1000 electrode with already optimum crystal density and orientation. Thus, the very limited electrocatalytic response towards NAD + reduction of the control electrode prepared by catalyst crystal growth without carboxyphenyl layer, as illustrated in Figure S2C, indicates that the density and orientation of the MOF crystals severely affect the catalytic performances. The XRD spectrum recorded for the grown NU-1000 film (Figure 1C) exhibits 4 peaks corresponding to the (010), (020), (021), and (040) crystal planes and fits well with the simulated spectrum. This provides further evidence that the NU-1000 film has been successfully generated on the GC surface.

The generated NU-1000 film plays the role of a host matrix for the immobilization of the actual Rh catalyst, and it is important to control its loading in order to tune both the conductivity and the electrocatalytic performance of the film. The strategy we used in this work for the S3). In a literature report about immobilization of ferrocenecarboxylic acid in MOF powder by the SALI approach, the ratio of ferrocene to Zr 6 has been approximately 1:1. [START_REF] Cai | Design Rules for Efficient Charge Transfer in Metal-Organic Framework Films: The Pore Size Effect[END_REF] In order to control the concentration of anchored Rh catalyst, we varied in our experiments the molar ratio of redox-a final molar ratio of 0.10, 0.58, and 0.91 for Rh atom /Zr node (Table S1), indicating that the surface concentration of the Rh catalyst follows the trend of its initial molar ratio with respect to the redox-inactive post-grafting agent, and more than 80% of the Zr 6 nodes can be in principle postfunctionalized with Rh-COOH. In order to confirm the distribution of the Rh catalyst inside the crystalline NU1000 rods, the NU1000 powder collected from the electrode was covered with resin and cut into thin sections. The cross-section of the NU-1000-Rh was observed by TEM, as shown in Figure 2A. Rh is homogeneously distributed, indicating that the Rh catalyst can be attached to the host matrix by post-functionalization, even inside the NU-1000 crystalline rods.

The presence of Rh catalyst inside the pores of NU-1000 was further confirmed by BET analysis (Figure 2B), because the total available surface area decreases from 2132 m 2 g -1 to 1917 m 2 g -1 , and the pore size is reduced from 3.68 nm to 3.38 nm after Rh catalyst loading as evidenced by DFT calculation (Figure 2C).

The electrochemical response of the GC-NU1000-Rh electrodes loaded with different ratios of Rh catalyst (0%,10%, 50% and 100%) were characterized by cylic voltammetry (CV) in 50 mM

Tris buffer at pH 7.2 under nitrogen. Tris buffer was chosen instead of PBS because NU-1000 is not stable in PBS buffer, as the phosphate ions coordinate with the NU-1000 framework, which has been actually used for drug release. [START_REF] Chen | Acid-Resistant Mesoporous Metal-Organic Framework toward Oral Insulin Delivery: Protein Encapsulation, Protection, and Release[END_REF] From the CV curves shown in Figure 3A, a peak corresponding to [Rh(Cp*)(bpy)Cl] + reduction is observed around -0.7 V, while the oxidation signal is located around -0.62 V. The amount of electrochemically active Rh catalyst was calculated by integration of the reduction peak in the cyclic voltammograms (Table S2).

Comparing these values with the Rh catalyst percentage estimated by EDS, it seems that they are following a similar trend as a function of the initial Rh catalyst ratio in the SALI solution (Figure S4A). We also tried to evaluate the average distance between the Rh centers for the different degrees of Rh loading based on a simplified geometric model (Figure S4B). The estimated distances are approximately 2nm for a sample with a nominal loading of 100%, 2.8nm for 50% and 5nm for 10%, indicating that even with the lowest loading, the redox centers are still close enough for allowing electron hopping.

However, the efficiency of the redox hoping being a function of the distance between the redox active center, the current increases for higher ratios of Rh catalyst in the NU-1000 film, and the electrode modified with 100% Rh-COOH (GC-NU1000-Rh) shows the highest redox signal.

Then, the electrocatalytic responses of GC-NU1000-Rh (0%, 10%, 50% and 100%) towards NAD + reduction was measured. is improved by up to two orders of magnitude compared to the value reported for a Rh catalyst functionalized on a CNTs-carbon felt electrode (0.11 A/cm 2 ), [START_REF] Zhang | Covalent Immobilization of (2,2′-Bipyridyl) (Pentamethylcyclopentadienyl)-Rhodium Complex on a Porous Carbon Electrode for Efficient Electrocatalytic NADH Regeneration[END_REF] indicating a much higher grafting density of the Rh catalyst per unit of active surface area on the GC-NU1000-Rh electrode and an efficient redox hoping throughout the MOF layer. [START_REF] Shen | Cerium-Based Metal-Organic Framework Nanocrystals Interconnected by Carbon Nanotubes for Boosting Electrochemical Capacitor Performance[END_REF][START_REF] Usov | Study of Electrocatalytic Properties of Metal-Organic Framework PCN-223 for the Oxygen Reduction Reaction[END_REF] . After having confirmed the tunable catalytic activity of such electrodes, another important aspect is to examine the possibility to use them for the quantitative regeneration of NADH.

Long-term experiments were therefore carried out with a GC-NU1000-Rh electrode, biased at a potential of -0.72 V in the presence of 0.5 mM NAD + under stirring (Figure 4A). After 5.8 h of electrolysis, spectra were used to characterize the produced 1,4-NADH, based on its intrinsic absorption peak at 340 nm. In order to make sure that the product is enzymatically active 1,4-NADH, and not some parasitic by-product, NAD-dependent alcohol dehydrogenase and acetaldehyde substrate were added to the solution, following a reported protocol [START_REF] Zhang | Bulk Electrocatalytic NADH Cofactor Regeneration with Bipolar Electrochemistry[END_REF] . The amount of produced 1,4-NADH was calculated from the decrease of the absorption at 340 nm (Figure 4B), based on a previously recorded calibration curve (inset in Figure 4B). These measurements we also used to study a potential leaching of the catalyst. Since the Rh catalyst has a typical absorption peak at 299 nm, 310 nm and 380 nm, [START_REF] Zhang | Covalent Immobilization of (2,2′-Bipyridyl) (Pentamethylcyclopentadienyl)-Rhodium Complex on a Porous Carbon Electrode for Efficient Electrocatalytic NADH Regeneration[END_REF] and we didn't observe an specific absorption signals at these wavelengths in the reaction product solution after long-term electrolysis, it is reasonable to assume that there is no leakage of Rh catalyst into the solution.

This was further confirmed by electrochemical measurements. The amount of electrochemically active Rh catalyst was calculated from the CVs, recorded before and after the electrocatalysis experiments. It decreases from 7.7610 -11 to 7.41x10 -11 mol, which corresponds to a loss of only 4%, indicating that there is no significant leaking of the incorporated Rh catalyst (Figure S6).

The amount of produced 1,4-NADH was estimated to be 6.36x10 -7 mol, corresponding to a Faradaic efficiency of 97%. The amount of electroactive [Rh(Cp*)(bpy)Cl] + on the electrode was estimated to be 7.7610 -11 mol by integrating the reduction peak of the CV. Thus, the turnover frequency (TOF) for NADH can be estimated to be ~1400 h -1 . Even though being lower than what can be achieved with an enzyme-based electroregeneration associated to surface immobilization [START_REF] Ganesan | Correlation between the Structure and Catalytic Activity of [Cp*Rh(Substituted Bipyridine)] Complexes for NADH Regeneration[END_REF] , it is nevertheless a high value for electrocatalytic NADH regeneration, compared to what has been reported so far in the literature for immobilized Rh complexes (3.6~1100 h -1 ). [START_REF] Zhang | Covalent Immobilization of (2,2′-Bipyridyl) (Pentamethylcyclopentadienyl)-Rhodium Complex on a Porous Carbon Electrode for Efficient Electrocatalytic NADH Regeneration[END_REF][START_REF] Ei-Zahab | Enabling Multienzyme Biocatalysis Using Nanoporous Materials[END_REF] This high efficiency can be ascribed to the precise loading and the homogeneous distribution of [Rh(Cp*)(bpy)Cl] + in the 3D framework, with a well-defined distance between each catalytic site (~2 nm estimated from the structure of NU-1000), ensuring that each catalyst unit experiences a similar microenvironment, which can efficiently take part in NADH regeneration.

Finally, the GC-NU1000-Rh electrode was used for electroenzymatic synthesis, and L-lactate dehydrogenase (LDH) has been chosen as a model enzyme for the conversion of pyruvate into Llactate. The size of LDH is reported to be 6 8.613.6 nm, [START_REF] Mondloch | Vapor-Phase Metalation by Atomic Layer Deposition in a Metal-Organic Framework[END_REF] and the pore size of NU1000-Rh is around 3.4 nm, thus LDH is not able to diffuse into the pores, but the small-sized NADH molecules (1.31.11.9 nm) [START_REF] Chen | Stabilization of Formate Dehydrogenase in a Metal-Organic Framework for Bioelectrocatalytic Reduction of CO 2[END_REF] can shuttle electrons between the Rh catalyst in the NU1000 film and LDH in the solution. In a chronoamperometry experiment with the GC-NU1000-Rh electrode biased at -0.72 V, in the presence of 0.1 mM NAD + and 200 U LDH, 1.6310 -6 mol pyruvate has been quantitatively converted into L-lactate after 9 h electrolysis, as characterized by HPLC (Figure S7). This corresponds to a TTN of ~21000 for the immobilized [Cp*Rh(bpy)Cl] + catalyst, whereas the highest TTN in the literature has been reported to be 12000 after reacting for 95h with an immobilized Rh catalyst [START_REF] Zhang | Electrocatalytic Biosynthesis Using a Bucky Paper Functionalized by [Cp*Rh(Bpy)Cl] + and a Renewable Enzymatic Layer[END_REF] . Thus, such a rather high value allows envisioning interesting applications in the frame of enzyme driven bioconversion.

CONCLUSION

We propose an original and highly efficient electrocatalytic system for NADH cofactor regeneration based on NU-1000 films immobilized on glassy carbon electrodes. An electrochemically grafted carboxyphenyl layer acts as an adhesion layer for the generated MOF films and helps controlling the density and orientation of the crystalline rods. The [Rh(Cp*)(bpy)Cl] + catalyst can be successfully immobilized at the Zr 6 nodes of the film via the SALI process. A precise loading of the catalyst is achieved by tuning the molar ratio between Rh catalyst and redox inactive species. The resulting electrocatalytic response towards NADH regeneration has been shown to be directly proportional to the amount of catalyst. The films with a maximum loading of Rh catalyst, corresponding to a final molar ratio of 0.9:1 with respect to the Zr 6 nodes, exhibit the highest electrocatalytic currents. Electrochemical bulk transformation experiments from NAD + to NADH reveal a very high TOF of ~1400 h -1 and an exceptionally high Faradaic efficiency of 97%, combined with an electroenzymatic conversion of pyruvate into L-lactate with a TTN of over 20000 when using L-Lactate dehydrogenase as a model enzyme.

This makes the proposed catalytic system very attractive for electroenzymatic synthesis, and also opens up interesting perspectives for generalizing the concept with other catalysts used in electroorganic synthesis.

Scheme 1 .

 1 Scheme 1. Schematic illustration of the elaboration process of a [Rh(Cp*)(bpy)Cl] +

Figure 1 .

 1 Figure 1. (A) XPS spectra of the C 1s signal for bare GC and the modified GC-COOH

  depicts SEM images of the obtained NU-1000 film, indicating that most of the crystals are growing in a perpendicular orientation to the electrode surface, and the length of individual rods is around 2~3 µm, as shown in the inset of Figure 1B. An optical picture of a representative GC-NU1000

Figure 2 .

 2 Figure 2. (A) TEM elemental mapping of the cross-section of a NU1000 crystal rod

Figure

  3B depicts the evolution of the response of a GC-NU1000-Rh electrode, and a linear increase of cathodic catalytic current was observed for successive additions of NAD + to the solution. The same experiments were also conducted with the electrodes functionalized with 0%, 10%, and 50% Rh catalyst (corresponding CVs are shown in FigureS5). Their CV response to the addition of 0.2 mM NAD + is shown in Figure3C, and their catalytic currents at -0.72 V as a function of NAD + concentration are shown in Figure 3D, indicating that the electrocatalytic performance towards NADH regeneration can be fine-tuned by a precise control the Rh catalyst loading. The resulting catalytic current density (16 A/cm 2 )

Figure 3 .

 3 Figure 3. (A) Cyclic voltammograms recorded with GC-NU1000-Rh electrodes with

Figure 4 .

 4 Figure 4. (A) Amperometric response recorded with a GC-NU-1000-Rh electrode in 0.5 mM NAD + at an applied potential of -0.72 V for 5.8 h in 30 mL Tris buffer (pH 7.2). (B)

catalyst fixation is based on solvent-assisted ligand incorporation (SALI). Carboxyl-

[Rh(Cp*)(bpy)Cl] + was incorporated at the Zr 6 nodes of the as-prepared NU1000 film by

dispersive X-ray spectroscopy (EDS). The different molar percentage (0%, 10%, 50%, and

100%) of the redox-active [Rh(Cp*)(bpy)Cl] + with respect to biphenyl-4-carboxylic acid lead to

ACKNOWLEDGMENT

The work has been funded by the National Natural Science Foundation of China (No. 21902045) and Henan center for Outstanding Overseas Scientist (GZS2020005). The project has also been supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement no 741251, ERC Advanced grant ELECTRA).

active Rh-COOH to inactive biphenyl-4-carboxylic acid during the SALI process. The obtained ASSOCIATED CONTENT Supporting Information.

Supplementary calculations of faradic efficiency and turnover frequency (TOF) for NADH regeneration and L-lactate production; Supplementary figures including cyclic voltammograms for different electrodes, optical pictures of the control electrode, atomic percentage measured from EDS, as well as calibration curves and HPLC measurements.

Corresponding Author

* lin.zhang@henu.edu.cn; *kuhn@enscbp.fr

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡ Weiwei Li and Chunhua Zhang contributed equally to this work.

Notes

The authors declare no competing financial interest.