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Covalent immobilization of (2,2’-Bipyridyl) (Pentamethylcyclopentadienyl)Rhodium Complex on a Porous Carbon Electrode for Efficient Electrocatalytic NADH Regeneration Lin Zhang, Neus Vilà, Gert-Wieland Kohring, Alain Walcarius, and Mathieu Etienne ACS Catal., Just Accepted Manuscript • Publication Date (Web): 17 May 2017 Downloaded from http://pubs.acs.org on May 17, 2017

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Covalent immobilization of (2,2’-Bipyridyl) (Pentamethylcyclopentadienyl)-Rhodium Complex on a Porous Carbon Electrode for Efficient Electrocatalytic NADH Regeneration Lin Zhang,a Neus Vilà,a Gert-Wieland Kohring,b Alain Walcarius,a and Mathieu Etiennea,* a

Laboratoire de Chimie Physique et Microbiologie pour l’Environnement (LCPME),

UMR7564 CNRS – Université de Lorraine, 405 rue de Vandoeuvre, F-54600 Villers-lèsNancy, France b

Microbiology, Saarland University, Campus, Geb. A1.5, D-66123 Saarbruecken, Germany. * Corresponding author: [email protected]

Abstract Covalent bonding of (2,2’-bipyridyl) (pentamethylcyclopentadienyl)-rhodium complex at the surface of a carbon-based porous electrode was achieved by combining diazonium electrografting, Huisgen cycloaddition and metal complexation. The immobilized catalyst was applied to electrochemical regeneration of the reduced form of nicotinamide adenine dinucleotide (NADH). The different steps of surface functionalization were characterized by X-ray photoelectron spectroscopy and electrochemistry. The Faradaic efficiency of NADH regeneration was 87 %. The chemical bonding provided good stability in solution under convection over 14 days, much better than the simple adsorption of the rhodium complex on the electrode surface. Finally, the system was tested in the presence of NAD-dependent 1 ACS Paragon Plus Environment

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dehydrogenases that were immobilized in a sol-gel film on the top of the functionalized porous carbon electrode. A total turnover of 3790 and turnover frequency of 164 h-1 was observed. Two enzymatic reactions were considered, D-fructose reduction to D-sorbitol catalyzed by D-sorbitol dehydrogenase and hydroxyacetone reduction to 1,2-propanediol with galactitol dehydrogenase. The electrocatalytic regeneration of 1 mM NADH by the immobilized rhodium species was kept after 90 h electrolysis in the conditions of electroenzymatic synthesis.

Keywords: electrosynthesis, ‘azide-alkyne’ click chemistry, rhodium catalyst, NADH regeneration, NAD-dependent dehydrogenase.

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Introduction Nowadays, NAD-dependent enzymes, especially dehydrogenases, are catalysts of increasing interest for enantioselective bioconversion of a variety of compounds, eg. alcohols, acids, and sugars.1 However, the practical applications are limited by the high price of the nicotinamide cofactors, so the regeneration of the latter is critical when considering a longterm biosynthesis system.2–6 The regeneration of cofactors can be basically realized by enzymatic,5,7 chemical,8,9 electrochemical10,11 or photoelectrochemical12,13 methods. Compared to chemical or enzymecoupled cofactor regeneration, which needs a second enzyme as well as a second substrate, electrochemical regeneration methods are getting rid of the side products from the coenzyme as well as the co-substrate separation. The main advantage of mass-free electrochemical regeneration method is to offer the possibility of simplifying bioconversion processes. In enzymatic reduction reactions, an intrinsically straightforward way for electrochemical regeneration of NADH would be the application of a negative potential to the electrode surface in order to reduce NAD+ back to NADH. However, the potential needed is very negative, -0.9 V vs SCE, as reported in the literature.14 At such cathodic value, NAD+ reduction will lead to NAD2 dimer instead of enzymatically active NADH, making the molecule unusable by the enzyme.1,15 Therefore, to prevent this dimerization at high overpotential, a catalyst who has a redox potential slightly more negative than the redox potential of cofactor NAD+/NADH (i.e., -0.56 V vs SCE), but not too low to avoid direct reduction of NAD+, is needed.16 In this respect, rhodium-based complexes [Cp*RhIII(L)Cl]+ proposed by Steckhan et al. are almost the only successful non-enzymatic regeneration catalysts for NADH regeneration.15,17–21 However, some reports show a degradation of the rhodium complex


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catalytic activity in the presence of amino acid residues22 or enzymes8,23 because –SH or – NH2 are reacting with the metal catalyst.24 Moreover, high concentration of the rhodium complex can also induce a decrease of the enzyme activity.8 Distancing the enzyme and the rhodium complex by immobilizing them separately provides better stability.22 Furthermore, immobilization of rhodium complex on electrodes will not only avoid the degradation of both chemical and enzymatic catalysts, but can also simplify the isolation of the reaction products. Two strategies have been previously developed to immobilize rhodium complexes on an electrode surface in an active form, as described in the following paragraphs. Both suffering from some limitations. The first approach is based on the generation of organic polymers bearing rhodium complexes deposited onto electrodes. Chardon-Noblat and coworkers prepared polypyrrolic rhodium(III) complex (2,2'-bipyridine or 1,10-phenanthroline ligand) films on electrode by anodic electropolymerization of pyrrole monomers.25 Beley and coworkers immobilized a rhodium(III) bis-terpyridinepyrrole complex by electropolymerization on a reticulated vitreous carbon electrode, studied the electrocatalytic reduction of NAD+ to 1,4-NADH, and applied it to the reduction of cyclohexanone to cyclohexanol in the presence of alcohol dehydrogenase.26 Finally, rhodium complexes were incorporated into the bilayers of vesicles formed from a electropolymerizable ammonium surfactant by a copolymerization reaction.27 Another polymerization route by ɤ-irradiation cross-linking was also reported to obtain [RhIII(C5Me5)(L)C1]+ complex polymer-modified electrodes.28 These methods provided a durable immobilization but suffer from dramatic loss of activity (e.g., only 26% electrochemically accessible22) that is mainly due to the steric shielding of the rhodium centers by the nonreactive polymer.


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The second strategy takes advantage of the π-π stacking between carbonaceous materials (e.g., graphene and carbon nanotubes) and aromatic moieties bearing the rhodium complex.29,30 This interaction favors a strong adsorption, yet noncovalent, of the rhodium complex on the carbon support. In this way, the electrode surface is covered with a monolayer of mediator, avoiding in principle the shielding effect aforementioned for thick polymer films, and the electron transfer between the rhodium complex and the electrode should be facilitated. Under these conditions, a surface-confined electrochemical behavior is expected.31 Park and coworkers reported the synthesis of a 3D-structured graphene–Rh-complex hydrogel, constituted of a phenanthroline modified Rh complex strongly immobilized on the graphene surface through π-π and π-cation interactions, demonstrating a repeated usability for electrochemical NADH regeneration up to seven cycles (2 h per cycle).29 Later on, Minteer and coworkers synthesized a rhodium complex with a pyrene-substituted phenanthroline ligand which was immobilized onto multi-walled carbon nanotubes via π-π stacking; a good catalytic response for the regeneration of NADH and repeated usability up to 10 cycles (30 min per cycle) were achieved.30 In both cases, the rhodium complex immobilization allowed the reusability of the catalyst over time, making it applicable in NADH-dependent enzymatic catalytic systems, but the method can be only applied to CNT- or graphene-based electrodes that have aromatic rings. On the other side, π–π stacking is a kind of reversible non-covalent bond, so expected to be less stable as a covalent one (ca. one order of magnitude difference in bond energy).32,33 In order to both taking advantage of a stable covalent binding and getting rid of electron shielding effects, up to now, attempt has been made to covalently immobilize thiolfunctionalized rhodium complex by self-assembled monolayers (SAMs) on gold electrodes.24 The molecules were electrochemically active but did not show any electrocatalytic properties with respect to NAD+ reduction. This inactivation phenomenon is probably due to unwanted 5 ACS Paragon Plus Environment

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coordination effects between thiol or amino groups and the rhodium metal center of the complex.8,23,24 A strategy to overcome this problem could be a step-by-step immobilization procedure, which would involve the incorporation of the rhodium metal center at the final step. This is what we have evaluated here through the covalent binding of bipyridine ligands to an electrode surface prior to forming the coordination compounds by reaction with pentamethylcyclopentadienylrhodium(III). The covalent immobilization of the [Cp*Rh(bpy)Cl]+ complex on porous carbon electrodes was achieved as illustrated in Scheme 1. The protocol involves 3 steps: (1) diazonium electrografting of an azido derivative, (2) Huisgen cycloaddition, also called “copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction”, to attach the bipyridine ligand, and (3) complexation with the rhodium species. The catalytic response of the immobilized complex was investigated with respect to the electrochemical regeneration of NADH from NAD+, and the long-term stability of this reaction was evaluated. Finally, the system was tested in the presence of redox enzymes that were immobilized in a sol-gel film on the top of such functionalized carbon electrode. Two enzymatic reactions were considered, D-fructose

reduction

to

D-sorbitol

catalyzed

by

D-sorbitol

dehydrogenase

and

hydroxyacetone reduction to 1,2-propanediol with galactitol dehydrogenase. The operational stability of the electrocatalyst was evaluated in both cases. Experimental Chemicals and Enzymes The following chemicals were used as received: tetraethoxysilane (TEOS, 98%, Alfa Aesar), 3-glycidoxypropyltrimethoxysilane (GPS, 98%, Sigma-Aldrich), β-nicotinamide adenine dinucleotide hydrate (NAD+, ≥ 96.5%, Sigma-Aldrich ), β-nicotinamide adenine dinucleotide, reduced disodium salt hydrate (NADH, ≥97%, Sigma-Aldrich), 4-azidoaniline hydrochloride 6 ACS Paragon Plus Environment

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(97%, Sigma-Aldrich), sodium nitrite (≥97.0% , Sigma-Aldrich), copper actate (Prolab), ascorbic

acid

(Prolab),

pentamethylcyclopentadienylrhodium(III)

chloride

dimer

((RhCp*Cl2)2, Sigma-Aldrich), D-fructose (99 wt%, Sigma), sodium dihydrogen phosphate (99.5%, Merck), hydroxyacetone (95%, Alfa Aesar), carbon felt (GFD 4.6 EA, density 0.09 g cm-3, electrical resistivity 3-6 Ω mm, Sigratherm®), poly(ethylene imine) (PEI, 50% w/v in water, Mn = 60000, Fluka), carboxylic-functionalized multi-walled carbon nanotubes (MWCNT, 95%, Φ 15±5 nm, L 1-5 µm, Nanolab). Overproduction of N-His(6) D-sorbitol dehydrogenase (800 U mL-1) and N-His(6)-Cysgalactitol dehydrogenase (60 U mL-1) were done with Escherichia coli BL21GOLD (DE3) containing the corresponding expression-vectors pET-24a(+) (Novagen) and purification of the enzyme was performed with Histrap columns (GE Healthcare) as described elsewhere.34 The protocol for synthesis of 4-[(2-propyn-1-yloxy)methyl]-4’-methyl-2,2’-bipyridine (alkynyl-bpy) is following a protocol reported in the literature (Scheme S1).24 The NMR spectroscopy data collected at each step are provided in supporting information. MWCNT-functionalized carbon felt electrode (CF-CNT) The surface of carbon felt was activated by recording 20 consecutive cyclic voltammetry scans from -0.7 V to 2.5 V in 0.1 M H2SO4 at a scan rate of 100 mV s-1. The carbon felt was then rinsed with water and subsequently heated up to 200℃ for 1h to remove the remaining H2SO4. Afterwards, the carbon felt was cut into pieces with dimension 0.5 cm * 0.5 cm. MWCNT-functionalized carbon felt was prepared by dipping the carbon felt into 0.5 mg mL-1 MWCNT suspension which is dispersed in an ultrasonic bath. The electrode was then dried in an oven at 130 ℃. The dipping/drying cycles were repeated 10 times. The electrode surface area was estimated from capacitance measurement to be around 1800 cm2. SEM characterizations of similar electrodes can be found elsewhere.35 7 ACS Paragon Plus Environment

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Azido-functionalized CF-CNT electrode (CF-CNT-Az) The diazonium cations were generated “in situ” by mixing 1 mM 4-azidoaniline and 2 mM sodium nitrite in 0.5 M HCl water solution with stirring for 5 min. The isolation of the diazonium salt is not required and grafting is performed using the same solution wherein diazonium cations were generated. The grafting process was carried out by electrochemical reduction achieved by cycling potentials in the range from 0.4 V to -0.6 V on a CF-CNT electrode (two consecutive cycles at a scan rate of 100 mV s-1). After electrografting, the electrode was rinsed with distilled water to remove the remaining ungrafted compounds. Covalent immobilization of bipyridine (bpy) on CF-CNT-Az electrode via Huisgen cycloaddition reaction (CF-CNT-bpy) A mixture of copper actate (1 mg) and ascorbic acid (2 mg) dissolved in an aqueous solution (3 mL) was added to a solution of alkynyl-bpy (2.4 mg) dissolved in dimethylformamide (7mL). The CF-CNT-Az electrode was immersed in this solution at room temperature for 24 h in darkness. After this period the electrode was rinsed carefully with water and DMF. Complexation reaction of rhodium with the CF-CNT-bpy electrode (CF-CNT-Rh) The ability of the bipyridine ligand to coordinate the transition metal was exploited in order to form the desired immobilized rhodium complex ([Cp*Rh(bpy)Cl]+). The CF-CNT-bpy electrode was immersed in a dichloromethane (20 mL) solution containing (RhCp*Cl2)2 (2 mg) under continuous stirring for 3h, and the resulting Rh complex was formed and immobilized onto the electrode surface thanks to the covalent bonding of bpy ligand. The electrode was then thoroughly washed with dichloromethane. For comparison purpose, an electrode with rhodium complex simply adsorbed was prepared by dipping a bare CF-CNT electrode in a 1


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mM [Cp*Rh(bpy)Cl]+ DMF/H2O (7/3) solution for 24 h, then following the same protocol as for the CF-CNT-Rh electrode (CF-CNT-adsorbed-Rh). Immobilization of enzymes An enzyme-containing silica sol was prepared according to a protocol that was previously described.34 Briefly, a mixture of 0.18 g TEOS, 0.13 g GPS, together with 0.5 mL water and 0.625 mL 0.01 M HCl was pre-hydrolyzed by stirring overnight. Then, this sol was diluted 3 times and a 200 µL aliquot was mixed with 100 µL of PEI (20%), 100 µL of water and 150µL DSDH (or GatDH) stock solution. This mixture was spread over the CF-CNT-Rh electrode and let to dry at 4°C before use. The CF-CNT-Rh-gel electrode was prepared by following the same protocol but replacing the enzyme solution with the same volume of water. Procedures and apparatus All electrochemical measurements were carried out using an Autolab PGSTAT-12 potentiostat, and performed in a sealed glass cell. A pencil core (0.5 mm diameter) glued to a copper wire by carbon black was used for connecting the carbon felt electrode. A Metrohm Ag/AgCl (3 M KCl) was used as reference electrode, and a steel bar served as auxiliary electrode. As oxygen reduction is observed in the working potential range, the solution was always purged with nitrogen for 15 min before performing electrochemical measurements and all the electrochemical characterizations were carried under nitrogen. For experiments requiring the addition of reagent aliquots (i.e., NAD+, D-fructose or hydroxyacetone additions), all mother solutions were purged with nitrogen before to be added into the cell with a syringe. Electrocatalytic activity was evaluated by cyclic voltammetry experiments with using a scan rate of 5 mV s-1. The amount, n, of rhodium complexes electrochemically active was estimated by integration of the charge, Q, involved in the cathodic redox peak. In our conditions, n=Q/2F, with F the Faraday constant. Electrosynthesis experiments were 9 ACS Paragon Plus Environment

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performed by chronoamperometry at a constant potential allowing the regeneration of NADH. This potential was chosen between -0.72 and -0.74 versus Ag/Ag/Cl according to the exact position of the redox peak from the rhodium electrocatalyst at the beginning of this experiment. UV-Vis spectra have been recorded on a Cary 60 Scan UV-Vis spectrophotometer. X-Ray Photoelectron Spectroscopy (XPS) analyses were performed using a KRATOS Axis Ultra Xray photoelectron spectrometer (Kratos Analytical, Manchester, UK) equipped with a monochromated AlKα X-ray source (hν = 1486.6 eV) operated at 150 W. The base pressure in the analytical chamber was 10− 9 mbar during XPS measurements. Wide scans were recorded using a pass energy of 160 eV and narrow scans using a pass energy of 20 eV (instrumental resolution better than 0.5 eV). Charge correction was carried out using the C(1s) core line, setting adventitious carbon signal (H/C signal) to 284.6 eV. Results and discussions Characterization of the alkynyl functionalized rhodium complex in solution This preliminary investigation is needed to ensure that the particular bipyridine derivative used here is likely to interact with rhodium-cyclopentadienyl moieties and that the resulting rhodium complex is indeed active towards the electrocatalytic reduction of NAD+. A bipyridine molecule was first functionalized with an alkynyl group (alkynyl-bpy) following a reported protocol,24 and its reaction with (RhCp*Cl2)2 was monitored by UV-visible spectroscopy. Curve a in Figure 1A displays the adsorption spectrum of the alkynyl-bpy compound alone in DMF/H2O (7/3) solution. A single absorption peak is observed at 282 nm which is ascribed to the π-π* electronic transitions of the bipyridine ligand. After 20 min in the presence of an excess amount of (RhCp*Cl2)2 in the solution, this peak at 282 nm decreased as a result of the complexation of the bipyridine ligand with the Rh metal center 10 ACS Paragon Plus Environment

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(the complexation process is illustrated in Figure 1B). The absorption spectrum of the complex is dominated by π-π* electronic transitions of the ligands which are however shifted to longer wavelengths, 299 nm and 310 nm, upon metal coordination. An additional weaker bands appears at 380 nm attributed to ligand-to-metal charge-transfer (LMCT) transitions (curve b, Figure 1A).36 Since the electrocatalytic activity of the rhodium complex has been proven to be influenced by the nature of functional groups attached to the bipyridine ligand,19,24,37 the complex functionalized with alkynyl group was first evaluated in solution (i.e., before immobilization) for NADH regeneration (Figure 2), and compared to the original rhodium complex reported in the literature ([Cp*Rh(bpy)Cl]+, see Figure S1). The experiment was performed at pH 6.5, i.e., in the optimal pH window determined previously.24 Figure 2 shows the electrochemical behavior of alkynyl-functionalized rhodium complex using a CF-CNT electrode in 50 mM PBS buffer. In the absence of NAD+, a reduction peak was observed at -0.680 V (see curve a on Figure 2), which was only shifted 5 mV more negative compared to the original compound (curve a, Figure S1B). This electrochemistry was poorly reversible as the anodic counterpart was ill-defined. Upon addition of NAD+ from 1 mM to 4 mM, an electrocatalytic current was observed, and the cathodic current was increasing proportionally to NAD+ concentration (see Inset in Figure 2). This variation of the electrocatalytic response to the addition of NAD+ with alkynyl functionalized rhodium was found similar to the evolution observed with unmodified [Cp*Rh(bpy)Cl]+ (see Figure S1). To conclude, the electrochemical catalytic properties of the alkynyl functionalized rhodium complex was not significantly influenced by the presence of the alkynyl moieties and could be therefore considered for the immobilization of the mediator on the electrode surface. Covalent immobilization of the rhodium complex on the electrode surface


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The covalent immobilization of the rhodium complex is achieved by the route illustrated in Scheme 1. The CF-CNT electrode is first functionalized with azide group by electrochemical reduction of the azidophenyl diazonium salt in situ generated from the 4-azidoaniline. Then the covalent bond between alkyne-functionalized bipyridine and azide moieties on the electrode (CF-CNT-Az) is formed through ‘azide-alkyne’ click chemistry, a bipyridinefunctionalized electrode (CF-CNT-bpy) is obtained. After complexation of with (RhCp*Cl2)2 in a last step, the desired rhodium complex immobilized onto the electrode (CF-CNT-Rh) is expected to be obtained. Each step is described and characterized hereafter. Step 1. Diazonium electrografting CF-CNT electrode was functionalized with azide groups by diazonium grafting. It was achieved by generating arylazide radicals from 4-azidoaniline and sodium nitrite in acidic medium, which can generate strongly reactive aryl radicals by electrochemical reduction that can in turn bind to CF-CNT. Here, the surface concentration of grafted organic groups had to be controlled, in order to avoid blocking of the electrode surface with an insulating layer. After optimizing the electrografting process, the modified surfaces were obtained by two reduction scans from 0.4 V to -0.6 V at a scan rate of 100 mV s-1 in 1 mM 4-azidophenyl diazonium cations, in order to obtain only a partial coverage of the surface. During the electrografting process, the first cyclic voltammogram exhibited an irreversible peak located around -0.3 V (see Figure S2A). During the second scan, the cathodic wave vanished and the reduction current decreased. This decrease was attributed to the presence of grafted azidophenyl groups on the electrode. The permeability of the organic layer was examined using ferrocenedimethanol as electroactive redox probe. The presence of the organic layer induced a limited decrease of the peak currents and only a slight increase of the ∆Ep between anodic and cathodic peaks from 0.23 V to 0.25 V (Figure S2B). This experiment proved that


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the electrode surface was effectively functionalized without preventing electron transfer reactions to occur. The attachment of the azidophenyl groups at the electrode surface was then evidenced by XPS. In addition to the characteristic C1s and O1s peaks, respectively at 285 eV and 532 eV, arising from the carbon substrate, the survey spectrum of the azido-functionalized electrode (not shown) also exhibited a N1s signal around 400 eV. The high-resolution XPS spectrum in the N1s region (Figure 3A) has been analyzed showing two main contributions, with a single peak at a binding energy of 404.1 eV and a larger and wider signal around 400 eV. An azide group is expected to present two peaks with a ratio 1:2 in this region.38 The central nitrogen, positively charged, exhibited a higher binding energy (404.1 eV) than the two other terminal nitrogen atoms (400.5 eV). However, a third contribution was also observed at 399.3 eV, and fitting the N1s region for the azido-modified substrate resulted in a ratio of 1:3.3, suggesting the presence of nitrogen species with a different environment in addition to the azide groups. This is likely due to the contribution of C-N=N-C bonds coming from the electrografting of the diazonium cations.39 Step 2. Huisgen cycloaddition The azido-functionalized electrode was then derivatized with bipyridine ligands via Huisgen cycloaddition, using alkynyl functionalized bipyridine in the presence of Cu(I) acting as a catalyst. This azide-alkyne coupling reaction leads to the formation of a triazole core substituted by a bipyridine ligand at the 4 position. Evolution of the high resolution nitrogen XPS spectra was employed to monitor the effectiveness of the chemical reaction. After quantitative click reaction, the triazole cores formed as a result of the azide-alkyne coupling and the incorporated bipyridine ligands should be the only species responsible of the nitrogen signal. The N1s spectrum (Figure 3B) displays 13 ACS Paragon Plus Environment

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a signal at 399.1 eV corresponding to uncharged species mainly attributed to the triazole cores and bipyridine ligands appearing at very close binding energies. A small peak at higher binding energies was observed indicating that some protonated or charged nitrogen atoms could be present. The possibility of remaining unreacted azide groups was discarded since the binding energies after the azide-alkyne coupling were shifted towards lower values. Analysis of the atomic concentration corresponding to each N1s from the area peaks indicates that only 15% of the starting azide functionalized groups have not been converted after click reaction. Step 3. Rhodium complexation The bipyridine–functionalized substrates were finally derivatized by coordination with (RhCp*Cl2)2. Successful complexation to form [Cp*Rh(bpy)Cl]+ moieties immobilized onto the electrode surface was pointed out by XPS and electrochemistry. The XPS survey spectrum proved the presence of Rh with the appearance of signals characteristic of Rh3d and Rh3p. As an illustration, Figure 3C displays the Rh3d core-level spectrum, showing a doublet at binding energies of 309.3 and 314.5 eV corresponding to 3d5/2 and 3d3/2, respectively. Cyclic voltammograms of the CF-CNT-bpy electrode before and after complexation with ((RhCp*Cl2)2) are shown in Figure S3. Before complexation, no redox peak was observed in the CV range from -0.4 to -0.9 V, which means that bipyridine itself is not electrochemically active in this potential window under the experimental conditions employed. After the formation of rhodium complex on the electrode (CF-CNT-Rh), a reduction peak was observed around -0.7 V, but no anodic signal was evidenced on a reversal scan. The absence of electrochemical reversibility has been reported before19,24 for the complex in solution and is explained by the protonation reaction described in Figure S1A. So the Rh(III) complex seems to be immobilized on the electrode surface with an electrochemical behavior close to that 14 ACS Paragon Plus Environment

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observed for similar species in solution (as reported before in Figure 2). We suppose that electron transfer reactions between the electrode surface and Rh(III) complexes is direct and that it does not go through the linking arm. The surface coverage was 0.6 pmol cm-2 (1.14 nmol molecules immobilized over a surface of 1800 cm2 estimated from capacitance of the electrode). The catalytic activity of CF-CNT-Rh electrode for NADH regeneration was then tested. Evaluation of electrocatalytic reduction of NAD+ Figure 4A depicts the evolution of the cyclic voltammetric response of CF-CNT-Rh electrode upon successive additions of NAD+ into the solution. As shown, the cathodic current measured at -0.7 V was increasing with the NAD+ content, but tended to level off when the final concentration of NAD+ reached 4 mM (see Inset of Figure 4A). This behavior is different from the one observed when the molecule was in solution, for which a linear increase of the current versus NAD+ concentration was observed in this concentration range (Figure 2). The saturation of the signal with the immobilized complex seems to be caused by the restricted amount of rhodium complexes available associated with limited heterogeneous electron transfer kinetics of the reaction.24 The rate constant of the reaction was here 0.66 ± 0.07 s-1. However, this behavior should not be a limitation in enzymatic-catalyzed electrosynthesis application as the cofactor NAD+ is used at low concentration, i.e. 1 mM or less. Under these conditions, the measured current would be only limited by the concentration and the mass transport of the cofactor (or the enzymatic substrate) in the porous electrode.35 After confirming the catalytic response, the functionalized electrode was applied to long-term electrochemical conversion (hours or days). A protective sol-gel layer was added at the surface of the CNT layer for limiting the loss of CNT from the CF-CNT electrode upon convective conditions, providing thereby a better mechanical stability to the composite


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material. This makes sense as such a sol-gel layer will be used later in the study to immobilize NAD-dependent dehydrogenases,35,40 in order to design a final bioelectrochemical system including these enzymes. Note that the presence of the gel layer does not prevent the electrocatalytic activity of the rhodium complex, showing that the catalytic current was similarly saturated for high concentrations of NAD+ (Figure S4). The rate constant was only reduced by a factor of 3 from 0.66 ± 0.07 s-1 to 0.21 ± 0.04 s-1, as a result of additional mass transport limitations. At first, we were wondering if the chemical attachment of the Rh complex really provided an advantage as compared to the simple adsorption of the molecule which was reported before.24 An electrode was prepared by simple adsorption of [Cp*Rh(bpy)Cl]+ onto CF-CNT for 24 h in DMF/H2O (7/3) solution, then covered with silica gel (CF-CNTadsorbed-Rh-gel). The stability of this CF-CNT-adsorbed-Rh-gel electrode was first compared with a CF-CNT-Rh-gel electrode (i.e., the electrode displaying the chemical bond between the carbon electrode and the rhodium complex). Both electrodes were introduced separately in 500 mL of 50 mM PBS buffer (pH 6.5) with a vigorous and continuous stirring. The electrochemical behavior of each electrode was repetitively tested with time. Figure 5A shows the evolution of the cathodic peak current attributed to the reduction of rhodium species measured for both electrodes; a different stability depending on the immobilization method employed in each case (either (a) covalently immobilized or (b) adsorbed rhodium complex) was observed. The CF-CNT-adsorbed-Rh-gel electrode had a relatively higher peak current at the beginning of the experiment. However, after one day in the buffer solution, the current dropped from 15.2 ± 0.5 µA to 5.3 ± 0.4 µA, which means that almost 63% of the adsorbed species were leaching out from the electrode to the solution. After 7 days, the peak current of the CF-CNT-ads-Rh-gel electrode was almost null. In comparison, the covalently immobilized CF-CNT-Rh-gel electrode had only 18% current decrease after one day. This 16 ACS Paragon Plus Environment

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ACS Catalysis

could be explained by desorption of a small amount of unreacted rhodium complex molecules remaining adsorbed on the surface after functionalization. The covalently immobilized CFCNT-Rh-gel electrode was then stable for at least 14 days in solution, with only 8% decrease of the catalytic current (with respect to that recorded after one day). This demonstrates unambiguously the interest of the covalent binding to improve the operational stability of the electrochemical response of the rhodium mediator. In addition to the reduction peak of rhodium complex, the stability of its catalytic activity was also confirmed by performing NAD+ electrochemical reduction experiments. Figure 5B shows the evolution of the catalytic current of the CF-CNT-Rh-gel electrode to the addition of NAD+ before and after running an electrolysis at -0.73 V (vs Ag/AgCl) for 39 h. The catalytic response to NAD+ remained almost unchanged. A similar experiment was performed after longer periods of time, i.e. 4 and 21 days (Figure S5). Some dissolution of the silica gel layer and some loss of CNTs was observed after 3 weeks stirring, but the saturated catalytic current in the presence of 3 mM NAD+ still reached 78% of the initial value (Figure S5). These tests indicate that the CF- CNT-Rh-gel electrode was stable in solution even under convective conditions, making it a suitable potential candidate for bioconversion processes in the presence of the enzymes. The main limitation was coming apparently from the stability of the electrode assembly more than from the chemical grafting of the mediator. The current efficiency of CF-CNT-Rh-gel electrode for NADH regeneration was evaluated by electrolysis (Figure 6). A potential of -0.73V was applied on the CF-CNT-Rhgel electrode and a catalytic current of 13 µA was observed upon addition of 0.5 mM NAD+ (Fig. 6A). An important question that arises concerns the selectivity of the electrochemical reaction, keeping in mind that NAD dimers and 1,6-NADH must be prevented. Exclusive regioselectivity was reported using [Cp*Rh(bpy)(H2O)]2+ for the reduction of natural NAD+


ACS Catalysis

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that provided 1,4-NADH14,19 and for the reduction of model NAD+ compounds.43 Enzymatically active 1,4-NADH is characterized by an absorption peak at 340 nm, while 1,6NADH has an absorption peak at 345 nm and dimeric species can show absorption on a wider range from 335 to 354 nm.41 The absorption peak measured at 340 nm supports strongly that NADH molecule was produced after 23 h electrolysis, even if confirmation of this result would involve more complex analytical protocols, such as NMR characterization of chromatographically isolated products.42,43 Considering that enzymatically active NADH is the product of the reaction, a concentration of 0.216 mM was estimated (Fig. 6B). This value is certainly underestimated because of the auto-catalytic degradation of NADH to NAD+ in this solution during the experiment. Note also that this measurement cannot be affected by the release of rhodium complex that adsorb in the same wavelength region because of the negligible amount of this molecule on the electrode surface. On the other hand, the theoretical concentration estimated from the charge involved in the electrocatalytic process led to a value of 0.248 mM NADH, corresponding to a Faradaic efficiency of 87%, largely higher than the quantitative values previously reported (23–36%) for immobilized rhodium complex on CNT by ‘π- π’ stacking.30 Considering the immobilization of 1.14x10-9 moles of rhodium complex on the electrode surface (determined by cyclic voltammetry), the total turnover was 3790 after 23 h, higher than the previously reported with Graphene–Rh-complex hydrogels.29 The turnover frequency (calculated by normalizing the production rate for NADH by the number of rhodium catalyst immobilized on the electrode) was 164 h-1. This value is one of the highest reported for the electrocatalytic reduction of NAD+ by immobilized rhodium complex26,28–30 (see table 1). Bioelectrocatalytic amperometric responses of electrodes with co-immobilized enzyme and redox mediator


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ACS Catalysis

After having confirmed the operational stability as well as the efficiency of the CFCNT-Rh-gel electrode for NADH regeneration, NAD-dependent dehydrogenases were introduced into the gel and both the rhodium complex and the enzyme were co-immobilized on the electrode. The bioelectrocatalytic response of CF-CNT-Rh-DSDH gel electrode was first demonstrated (Figure 7A) by injecting D-fructose substrate into 50 mM PBS buffer containing 1mM NADH cofactor. DSDH reduces D-fructose to D-sorbitol and consumes NADH to generate NAD+, which can then be reduced by the rhodium complex at the electrode surface to regenerate NADH for further enzymatic transformation (this electrochemical cycle is shown in Figure S6). The apparent Km estimated from the chronoamperometric experiment was 0.4 mM, much lower than the Km of free DSDH in buffer solution (Km is 49.4 mM).44 The electrocatalytic reduction was evaluated after 25 and 90 h of electrolysis in the presence of DSDH (see Figure S7). A decrease of the maximum electrocatalytic current was observed with time, but comparable electrocatalytic currents were observed in the presence of 1 mM NAD+ (i.e. the concentration used in the electroenzymatic experiment) before starting the electrolysis experiment (12.7 ± 4.8 µA), after 25h electrolysis (10.3 ± 0.9 µA ) and after 90h electrolysis (11.1 ± 3.5 µA). The system is thus promising for integration in a bioelectrochemical reactor for long-term bioconversion experiments, but great care has to be taken in order to separate even more efficiently proteins and rhodium complexes. It can moreover be extended to any NAD-dependent proteins, as shown here with galactitol dehydrogenase (GatDH). GatDH is a multifunctional enzyme that has a broad substrate spectrum, for example involved in the production of rare sugar for medicine production.45 Figure 7B shows the amperometric response recorded upon successive additions of hydroxyacetone substrate in 50 mM PBS buffer containing 1 mM NADH cofactor and 1 mM MgCl2 on a CNT-Rh-GatDH gel electrode (which contained GatDH instead of DSDH). The electrode response was lower than in previous experiment with DSDH


ACS Catalysis

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due to a lower enzymatic activity of GatDH (1.8 U GatDH immobilized on the electrode versus 24 U DSDH in the previous experiment). An apparent Km of 0.2 mM was estimated for this protein in the bioelectrode, indicating again a high affinity of the immobilized enzyme to its substrate hydroxyacetone.46

Conclusion [Cp*Rh(bpy)Cl]+ was successfully immobilized on a porous carbon electrode by covalent binding. Azido-functionalized electrodes were first prepared by electrochemical reduction of 4- azidophenyl diazonium cations that were used later on to attach bipyridine ligands via cycloaddition Huisgen reaction. A final post-functionalization step was based on the ability of bipyridine ligands to coordinate with rhodium metal centers such as (RhCp*Cl2)2 dimer under mild conditions. The long-term stability of this rhodium complex functionalized electrode towards NAD+ reduction was evaluated and showed a high Faradaic efficiency of 87%. The rhodium complex functionalized electrode was successfully combined with D-sorbitol dehydrogenase and galactitol dehydrogenase. In those conditions the electrocatalytic regeneration of 1 mM NAD+ by the immobilized rhodium species was kept very effective, even after 90 h electrolysis, making this system promising for application to electroenzymatic synthesis. Acknowledgements The authors are grateful to ICEEL for the financial support through the project BIOCARBON (n° 061-PT). Lin Zhang gratefully acknowledges CNRS and Region Lorraine for PhD funding. We also thank Aurelien Renard (LCPME) for XPS measurements. Supporting information


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ACS Catalysis

Synthesis route and NMR spectroscopy data corresponding to the molecule at each step, schemes of the electrocatalytic and enzymatic reactions and additional electrochemical data (electrocatalytic regeneration of NADH by [Cp*Rh(bpy)Cl]+ in solution, electrografting experiment and electrochemical characterization with ferrocenedimethanol, characterization of the complexation reaction, electrocatalytic reduction of NAD+ in the presence of silica gel, catalytic currents after 4 days and 21 days, electrocatalytic reduction of NAD+ in the presence of enzyme, after 25 and 90 h electrolysis at -0.74 V vs Ag/AgCl.

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ACS Catalysis

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Cp*

Cl Rh N

N

N

N

N

N

N

+

N

N

O

-

N N N

NH2

+

N N

-

O

Rh Cl Cl Cl Cl Rh

O

N

N N

N

Cp* =

Step 2. CuAAC click reaction

Step 1. Diazonium electrografting

CF-CNT

CF-CNT-Az

-

Step 3. Complexation

CF-CNT-Bpy

CF-CNT-Rh

Scheme 1. Synthetic route followed for the functionalization of a carbon electrode surface with [Cp*RhIII(bpy)Cl]+.


Page 25 of 33

A

B 2.0

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

a

1.5

Cl

N

N

CH 3

2

CH 3

2 O

O

0.5

Rh N

N

b

1.0

+

Rh Cl Cl Cl Cl Rh

+ 2 Cl-

0.0

CH

CH

300

400

500

a

b

Wavelength / nm

Figure 1. (A) UV-vis spectra recorded for alkynyl-bpy in DMF/H2O (7/3) solution (a) before and (b) after complexation in the presence of a molar excess of (RhCp*Cl2)2. (B) Corresponding illustration of the complexation process.


ACS Catalysis

a b

-0.4

c

Icat / mA

0.0

I / mA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 33

0.4 0.2 0.0 0

2

4

C / mM

d e

-0.8

from 0 to 4 mM NAD -0.8

-0.6

+

-0.4

E / V vs. Ag/AgCl

Figure 2. Cyclic voltammograms recorded at a potential scan rate of 5 mV s-1 using a CFCNT electrode in a solution containing 0.1 mM alkynyl functionalized rhodium complex, with gradually added NAD+: (a) 0 mM; (b) 1 mM; (c) 2 mM; (d) 3mM; (e) 4 mM. Experiments were carried out in 50 mM PBS (pH 6.5) solution, under nitrogen. Inset is the variation of the catalytic current versus the concentration of NAD+. The geometric surface area of the electrode was 0.25 cm2.


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ACS Catalysis

-N=N+=N-

A

-N=N+=N-C-N=N-C

408

404

400

396

392

Binding energy / eV

B

408

404

400

396

392

Binding energy / eV

C

320

318

316

314

312

310

308

306

304

Binding energy / eV

Figure 3. (A,B) XPS spectra of the N1s region obtained from the azido-functionalized substrate, respectively before (A) and after (B) ‘click’ reaction with the bipyridine ligands. (C) XPS Rh3d core-level spectrum of a CF-CNT-Rh electrode.


ACS Catalysis

A

B

NAD+

100

NADH +

0

no NAD

-100 c

-200 -300

e f

d

b

+ Cl Rh

a N

150 100 50 0

Icat / µA

I / µA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 33

4 mM NAD

+

N

O N

0

2

4

C / mM

N N

2e-0.8

-0.6

-0.4

E / V vs. Ag/AgCl Figure 4. (A) Cyclic voltammograms recorded at a potential scan rate of 5 mV s-1 using a CFCNT-Rh electrode in 50 mM PBS buffer (pH 6.5), under nitrogen, to gradually added NAD+: (a) 0 mM; (b) 0.5 mM; (c) 1 mM; (d) 2 mM; (e) 3 mM; (f) 4 mM. (B) Schematic representation of NADH regeneration mediated by a CF-CNT-Rh electrode. The geometric surface area of the electrode was 0.25 cm2.


Page 29 of 33

30

A

15 12 9

a

6 3

b

0 0

2

4

6

8 10 12 14

Catalytic current / µA

18

Catalytic current / µA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

B

25

a

20

b

15 10 5 0 0

+

[NAD ] / mM

1

2

3

4

5

+

[NAD ] / mM

Figure 5. (A) Evolution of the reduction catalytic currents measured from CV in a solution containing 0.5 mM NAD+, using (a) CF-CNT-Rh-gel and (b) CF-CNT-adsorbed-Rh-gel electrodes. (B) Catalytic currents measured from CV versus NAD+ concentration using a CFCNT-Rh-gel electrode (a) before and (b) after running a 39 h period at a potential of -0.73 V in the presence of 1 mM NAD+. Other conditions as in fig. 4.


ACS Catalysis

0.5 mM NAD

Absorbance

-4

3

A

+

-6 -8 -10 -12

B

Absorbance

-2

I / µA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 33

2

3 2

340 nm

1 0 0.0 0.2 0.4

[NADH] / mM 1

-14 -16

0 0

5

10

15

t/h

20

300

350

400

Wavelength / nm

Figure 6. (A) Electrolysis current measured in the presence of 0.5 mM NAD+ , as a function of time, using a CF-CNT-Rh-gel electrode at an applied potential of -0.73 V in 20 mL 50 mM PBS buffer (pH 6.5), under nitrogen. (B) Absorbance signal measured after 23 h electrosynthesis. Inset: calibration curve of NADH in 50mM PBS buffer (pH 6.5), the square point correspond to the NADH formed from NAD+ after 23 h. The geometric surface area of the electrode was 0.25 cm2.


0

-2

A b

-I / µA

I / µA

-20 -30

40 30 20 10

b

-4 -5 -6

a

0 1 2 3

C / mM

0

B

-3

-10

I / µA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Catalysis

-I / µA

Page 31 of 33

6 5 4

C / mM

-7

1

2

3

a

01234

0

t/h

20

40

t / min

Figure 7. (A) Amperometric responses to increasing concentrations of D-fructose (from 0.5 to 3 mM) recorded using (a) CF-CNT-Rh-DSDH-gel electrode and (b) CF-CNT-Rh-gel electrode, as recorded at an applied potential of -0.74 V in 50 mM PBS (pH 6.5) containing 1 mM NADH. (B) Amperometric responses to increasing concentrations of hydroxyacetone (from 1 to 4 mM) recorded using (a) CF-CNT-Rh-GatDH-gel electrode and (b) CF-CNT-Rhgel electrode, as recorded at an applied potential of -0.72 V in 50 mM PBS (pH 6.5) containing 1 mM NADH and 1mM MgCl2. The geometric surface area of the electrode was 0.25 cm2.


ACS Catalysis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Table 1. Comparative performance summary of immobilized rhodium-based complexes with respect to NADH regeneration.

Mode of immobilization on the electrode

TONa

N-vinyl-pyrrolidone modified electrode with pendant rhodium complexes Bonding of the rhodium complexes on ω214 functionalized amino polyethylene glycol Polypyrrole rhodium bis-terpyridine modified Up to 113 electrode Graphene–Rh-complex hydrogels 1 000 Rhodium complex immobilized on multiwalled carbon nanotubes via π-π stacking Covalently-bound rhodium complexes on 3790 carbon electrode a TON, turnover number; b TOF, turnover frequency.

TOFb

Ref.

20 h-1 at 25 °C 67.2 h-1 at 38 °C

28

-

22

-

26

57 h-1

29

3.6 s-1

30

164 h-1

this work


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ACS Catalysis

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