A Pyrene‐Triazacyclononane Anchor Affords High Operational Stability for CO2RR by a CNT‐Supported Histidine‐Tagged CODH

Abstract An original 1‐acetato‐4‐(1‐pyrenyl)‐1,4,7‐triazacyclononane (AcPyTACN) was synthesized for the immobilization of a His‐tagged recombinant CODH from Rhodospirillum rubrum (RrCODH) on carbon‐nanotube electrodes. The strong binding of the enzyme at the Ni‐AcPyTACN complex affords a high current density of 4.9 mA cm−2 towards electroenzymatic CO2 reduction and a high stability of more than 6×106 TON when integrated on a gas‐diffusion bioelectrode.


Materials and methods
Materials and Instruments. 1-pyrenebutyric acid adamantyl amide was prepared as previously described. [1] All reagents were purchased from Sigma Aldrich. Commercial grade thin Multi-Walled Carbon Nanotubes (MWCNT, 9.5 nm diameter, purity > 99%). Carbon nanomaterials were used as received without any purification. RecRrCODH was co-produced in presence of the three Ni-chaperones (RrCooC, RrCooT and RrCooJ) and isolated as previously described [2] . When not used, the enzymes were stored at 4 °C. All the reagents were used without further purification. All solvents were of analytical grade. Distilled water was passed through a Milli-Q water purification system. Acetonitrile (HPLC) grade used for electrochemistry was obtained from VWR chemicals and used after drying on 4 Ȧ molecular sieves. NMR spectra were recorded on a Bruker AM 300 ( 1 H at 300 MHz, 13 C at 75 MHz) or a Bruker Avance 400 ( 1 H at 400 MHz, 13 C at 100 MHz). Chemical shifts are given relative to solvent residual peak. Mass spectra were recorded on a Bruker Esquire 3000 (ESI/Ion Trap) equipment.

Electrochemical analysis.
The electrochemical experiments in aqueous media were performed in 50 mM TrisHCl buffer pH 8.5 in a three-electrode electrochemical cell, using a Biologic VMP3 Multi Potentiostat, inside an anaerobic glove box (O2 <2 ppm, Jacomex). The surface of GC electrodes was polished with a 2 μm diamond paste purchased from Presi (France) and rinsed successively with water, acetone, and ethanol. A Pt wire placed was used as counter electrode, and the SCE or Ag/AgCl served as reference electrodes. All current densities are given considering the geometrical surface of the MWCNT-modified electrode (0.07 cm -2 ). Oxygen concentrations were measured in the electrolyte by using a Neofox Oxygen Sensing System from OceanOptics.

Synthetic procedures for compound 1 and 2:
Addition of 1-Pyrenylmethyl bromide (555mg, 1.87mmol) to a solution of tacn orthoamide (260 mg, 1.87 mmol) in tetrahydrofuran (6 ml) produced a precipitate almost immediately. Stirring was continued for another 30 minutes after which the product was filtered and washed with absolute ethanol (2 x 2 ml) and ether (3 x 2 ml). The green solid was then dissolved in water (6 mL) and heated at reflux for 4 hours. The solution pH was adjusted to 12 with NaOH, the product was extracted into chloroform (4 x 10 ml), the extracts dried over magnesium sulfate and the solvent removed under reduced pressure to give 1 (411 mg, 87 % yield). 1 (411mg, 1.1 mmol) was then dissolved in acetonitrile (30 mL), sodium carbonate (3 g) and ethyl bromoacetate (252 µL, 2.3 mmol) were added. The mixture was stirred at reflux for 6 h. Solvents were removed under reduced pressure. The crude product was dissolved in a 5M aqueous solution of NaOH (pH=12) extracted into chloroform (3 x 10 ml). The gathered extracts were dried over magnesium sulfate and the solvent removed under reduced pressure to give a viscous, yellow/orange oil. The product was purified by column CH2Cl2/Acetone affording a white powder (255mg, 63% yield). 1

Synthetic procedures for AcPyTACN:
Compound 2 (225m g 0.49 mmol) was dissolved in 5M HCl (5 ml) and the solution refluxed for 3h. Removal of the solvent gave the deprotected AcPyTACN as a green solid.

Preparation of the electrodes
The working electrodes were glassy carbon and gas diffusion electrodes (  Dithionite (DTH) was diluted to 23 nM (monomer) in 50 mM TrisHCl pH 8.5, remaining 1 µM of DTT and DTH. Then, the enzyme was exposed to the air for several times (0-40 minutes) at 25 °C.
The enzyme was further diluted to 5 nM (monomer) in anaerobic 50 mM TrisHCl pH 8.5, 5 mM dithiothreitol (DTT), 1 mM dithionite (DTH) and incubated 5 minutes in this buffer in order to preactivate the enzyme before measuring the remaining specific activity.

ICP-AES metal ion analysis
MWCNT films of 1.8 cm -2 were modified according to the procedure described in previous section.
Then, the modified MWCNT electrodes were mineralized in the presence of 0,6 mL HNO3 (65%) at 60 °C for 24 h. In order to remove traces of carbon nanotubes, the solution is first centrifuged 10 min at 5000 rpm, filtered on a glass filter and washed with 10% nitric acid solution before completing the volume to 6ml with pure water. . The metal concentration of the supernatant was analyzed by inductively coupled plasma atomic emission spectroscopy (ICP-AES) (Shimadzu ICP 9000 with mini plasma torch in axial reading mode). Standard solutions of Ni and Fe for atomic absorption spectroscopy (Sigma Aldrich) were used for quantification (calibration curve between 1.9 and 1000 μg L −1 in 10% HNO3 (Fluka)). The results are presented in Table S1 Table S1. ICP-AES metal ion analysis (Concentration in μM in the supernatants)

Electrochemical analysis
The Langmuir-Freundlich model was employed to fit the experimental data using OriginPro 2020, according to equation 1: [3,4] Where %Losseq is the percentage of CO activity loss at the equilibrium, %lossmax is the maximum percentage of CO activity loss at maximum imidazole concentration, KImid app is the apparent association constant in water between imidazole and AcPyTACN sites at the modified electrode and n is Langmuir-Freundlich coefficient number. Table S2 shows the Langmuir-Freundlich model parameters obtained from fitting curves from Figure 4A.