Visible light-induced reduction system of diphenylviologen derivative with water-soluble porphyrin for biocatalytic carbon–carbon bond formation from CO₂

Materials

Tetraphenylporphyrin tetrasulfonate (H₂TPPS) was purchased from Dojindo Laboratories (Kumamoto, Japan). Zinc acetate dihydrate, methanol, acetonitrile, sodium sulfanilate dihydrate, sodium hydrosulfite and triethanolamine (TEOA) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 4,4′-Bipyridyl, 1-chloro-2,4-dinitrobenzene, PV²⁺ and MV²⁺ were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). ME from chicken liver (EC 1.1.1.40) was purchased from Sigma-Aldrich Co. LLC. All other materials were of analytical grade or the highest grade available. Zinc tetraphenylporphyrin tetrasulfonate (ZnTPPS) was synthesized according to the previous reported method [34], [35].

Preparation of 1,1,′-bis(p-sulfonatophenyl)-4,4′-bipyridinium dichloride (PSV²⁺)

Synthesis route of PSV²⁺ was summarized in Fig. 5. First, 1,1′-bis(2,4-dinitrophenyl)-4,4′-bipyridinium dichloride was synthesized according to a published procedure [36] as follows. 4,4′-Bipyridyl (1.6 g, 10 mmol) and 1-chloro-2,4-dinitrobenzene (7.1 g, 35 mmol) were dissolved in anhydrous acetonitrile (30 mL). The solvent was stirred at 90°C for 72 h under reflux. After cooling to room temperature, the mixture was diluted with acetonitrile and filtered to obtain yellow-white product. Then product was dried under vacuum overnight. Next, PSV²⁺ was synthesized according to the previous reported method [37] with modified as follows. 1,1′-Bis(2,4-dinitrophenyl)-4,4′-bipyridinium dichloride (1.1 g, 2.0 mmol) was dissolved in distilled water (40 mL) and a solution of sodium sulfanilate dihydrate (2.1 g, 9.0 mmol) in distilled water (50 mL) was added dropwise. The mixture was stirred at 20°C for 24 h and refluxed at 80°C for 48 h. The red solvent was evaporated, and then dark red solid was obtained as PSV²⁺.

Fig. 5:

Synthesis scheme of 1,1′-bis(p-sulfonatophenyl)-4,4′-bipyridinium dichloride (PSV²⁺).

Fluorescence quenching behavior of ZnTPPS by PSV²⁺

Quenching of photoexcited state of ZnTPPS by PSV²⁺ was investigated using steady state fluorescence spectroscopy. The sample solution containing ZnTPPS (0.5 μM) and PSV²⁺ in 10 mM bis-tris buffer (pH 7.4). The concentration of PSV²⁺ was varied from 0 to 50 μM. The excitation wavelength was 422 nm due to the Soret band of ZnTPPS. The fluorescence emission spectrum of ZnTPPS was measured using a fluorescence spectrophotometer (SHIMADZU, RF-5300PC). In addition, quenching of photoexcited state ZnTPPS by PV²⁺ also was studied as a reference.

Spectroscopic properties of the reduced form of PSV²⁺

The UV-visible absorption spectra of one- and two-electron reduced form of PSV²⁺ were measured. The one- and two-electron reduced form of PSV²⁺ were reduced as follows. PSV²⁺ solution was prepared with bis-tris buffer (pH 7.4) in the cell and 2 mM of sodium hydrosulfite was add to the sample solution. Then, two-electron reduced form of PSV²⁺ was produced and oxidized to one-electron reduced from of PSV²⁺ by oxygen. The one- and two-electron reduced form of PSV²⁺ were detected by UV-visible absorption spectroscopy (SHIMADZU, MultiSpec-1500).

Reduction potential measurement of PSV²⁺

For the electrochemical properties of PSV²⁺, the reduction potentials were measured by cyclic voltammetry (HOKUTO DENKO, HZ-3000) used a glassy carbon electrode as a working electrode, a platinum electrode as a counter electrode, and a Ag/AgCl electrode as a reference electrode. The electrolyte solution was nitrogen-saturated 0.2 M KCl solution. The scan rate was adjusted to 100 mV min⁻¹. In addition, the reduction potentials of PV²⁺ also were measured as a reference.

Photoreduction of PSV²⁺ by photosensitization of ZnTPPS

The sample solution was consisted of TEOA (0.3 M), ZnTPPS (10 μM) and PSV²⁺ (0.1 mM) in 5.0 mL of 10 mM bis-tris buffer (pH 7.4). The sample solution was deaerated by freeze-pump-thaw cycles repeated 6 times. The sample solution in the cell equipped with a magnetic stirrer was irradiated with a 250 W halogen lamp (TOSHIBA) with light intensity of 200 Jm⁻²s⁻¹ at 30°C. Ultraviolet ray with wavelength of shorter than 390 nm were blocked with cut-off filter. The reduced form of PSV²⁺ production was monitored by UV-visible absorption spectroscopy (SHIMADZU, MultiSpec-1500).

Photoinduced malate production from CO₂ and pyruvate with ME and PSV²⁺

Visible light-induced malate production from CO₂ and pyruvate with TEOA, H₂TPPS, PSV²⁺, Mg²⁺ and ME was carried out as follows. A sample solution containing TEOA (0.2 M), H₂TPPS (40 μM), PSV²⁺ (0.4 mM), sodium pyruvate (12 mM), MgCl₂ (10 mM) and ME (4.0 units; 0.93 μM) in 5 mL of 10 mM bis-tris buffer (pH 7.4) was deaerated by freeze-pump-thaw cycles repeated 6 times, and then flushed with CO₂ gas for 10 min. The sample solution in the cell equipped with a magnetic stirrer was irradiated with a 250 W halogen lamp with light intensity of 200 Jm⁻²s⁻¹ at 30°C. Ultraviolet ray with wavelength of shorter than 390 nm were blocked with cut-off filter. The concentration of oxaloacetate and malate production in sample cell were analyzed by an ionic chromatograph system (Thermo Fisher Scientific, Dionex IC-1100). For the experiment of visible light-induced malate production, zinc acetate is added in excess amount for the ZnTPPS synthesis, so excess amount of zinc ion is remained. Zinc ion is a divalent ion, which affects the activity of the ME, so metal-free porphyrin (H₂TPPS) with little difference from the photosensitization activity of ZnTPPS was used as a photosensitizer. In this condition, it is confirmed that Mg²⁺ is not coordinated to H₂TPPS by using UV-visible absorption spectroscopy.

Fluorescence quenching behavior of ZnTPPS by PSV²⁺

The interactions between ZnTPPS and PSV²⁺ were studied by measurement of the fluorescence of ZnTPPS in the presence of PSV²⁺. Figure 6 shows the fluorescence spectrum change of ZnTPPS with PSV²⁺ concentration. The fluorescence maximum of the ZnTPPS at 606 and 656 nm decreased with an increasing PSV²⁺ concentration. This result indicated that the fluorescence from excited singlet state of ZnTPPS was quenched by PSV²⁺.

Fig. 6:

Fluorescence spectrum change of ZnTPPS with addition of PSV²⁺. The sample solution consisted of ZnTPPS (0.5 μM) and PSV²⁺ in 10 mM bis-tris buffer (pH 7.4). The excitation wavelength was 422 nm.

Figure 7 shows the relative fluorescence intensity change with PSV²⁺ concentration (Stern-Volmer plot [38], [39]). The ratio of fluorescence intensities of ZnTPPS in the absence of Quencher (Q) and the presence of Q (I₀/I) increased linearly.

Fig. 7:

Stern-Volmer plot for the fluorescence quenching of ZnTPPS by PV²⁺ (●), PSV²⁺ (■). The excitation and fluorescence wavelengths were 422 and 606 nm, respectively.

I0/I=1+KSV[Q]

I₀ and I are the fluorescence intensities in the absence and the presence of Q. K_SV is the Stern-Volmer quenching constant (M⁻¹), and [Q] is quencher concentration (M⁻¹). The K_SV value is obtained from the slope of I₀/I versus [Q]. The K_SV values of PSV²⁺ and PV²⁺ to ZnTPPS were obtained to be 4.9×10³ and 8.3×10⁴ L mol⁻¹, respectively. The K_SV value of PSV²⁺ was smaller than that of PV²⁺. PSV²⁺ is anion with sulfonate-group and electrostatically repulsive with ZnTPPS with sulfonate-groups. The interaction between ZnTPPS and PSV²⁺ was smaller than that between ZnTPPS and PV²⁺.

Reduction potentials for PSV²⁺

The reduction potentials of PSV²⁺ and PV²⁺ are listed in Table 1. The first and second reduction potentials of PV²⁺ derivatives were determined by cyclic voltammetry using an Ag/AgCl electrode as a reference electrode. The first and second reduction potentials of PV²⁺ were estimated to be −0.39 and −0.74 V, respectively. On the other hand, the first and second reduction potentials of PSV²⁺ were estimated to be −0.38 V and −0.72 V, respectively. Compared to the reduction potentials of PV²⁺ and PSV²⁺, there are little difference, so it is considered that the sulfo-group has little effect on the reduction potential in PV²⁺. In addition, reduction efficiency of 4,4′-bipyridinium derivatives with photosensitization of ZnTPPS depends on the reduction potential of 4,4′-bipyridinium derivatives [21]. Therefore, it is considered that both PSV²⁺ and PSV⁺ are reduced with the ZnTPPS as well as that using PV²⁺. PV⁺ and PSV⁺ also have reducing ability of oxaloacetate to malate.

The reduction potentials of PVs were measured by cyclic voltammetry with glassy carbon (GC) as a working electrode, Pt as a counter electrode and Ag/AgCl as a reference electrode in 0.2 M KCl aqueous solution.

Spectroscopic properties of the reduced form of PSV²⁺

Figure 8 shows the UV-visible absorption spectra of one- and two-electron reduced form of PSV²⁺ in absorbance baseline was oxidation form of PSV²⁺ solution, which had a yellow color solution. When the PSV˙⁺ was formed, the color of solution was changes to dark orange, and it had an absorption band with the maximum 490 nm (Curve 1). In addition, when PSV⁰ was produced, the solution changed to green, and it had an absorption band with the maximum 650 and 710 nm, respectively (Curve 2).

Fig. 8:

UV-visible absorption spectra of one-electron reduced form of PSV²⁺ (PSV˙⁺, curve1) and two-electron reduced form of PSV²⁺ (PSV⁰, curve2).

Photoreduction of PSV²⁺ with visible light sensitization of ZnTPPS

Figure 9 shows difference UV-visible absorption spectrum change with visible light irradiation in a reaction system consisting of TEOA, ZnTPPS and PSV²⁺. The absorption band at 650 and 710 nm due to the PSV⁰ was increased with irradiation time. The color of the sample solution changed to pale green due to the PSV⁰ production. Thus, two-electron reduction of PSV²⁺ to PSV⁰ was proceeded with visible light sensitization of ZnTPPS in this system. The photoinduced electron transfer from ³ZnTPPS* to PSV²⁺ was proceeded in this system. The redox potentials of ³ZnTPPS*, E(ZnTPPS⁺/³ZnTPPS*) and E(³ZnTPPS*/ZnTPPS⁻) were reported to be −0.75 and 0.45 V (vs. Ag/AgCl), respectively [34]. The first and second reduction potentials of PSV²⁺ were estimated to be −0.38 and −0.72 V, respectively. Figure 10 shows the redox potential diagram of ZnTPPS and PSV²⁺. As the Gibbs free energy for the photoinduced electron transfer from ³ZnTPPS* to PSV²⁺ to produce the PSV⁰ was estimated to be −5.8 kJ mol⁻¹, thus this process is thermodynamically possible reaction. The oxidation potentials of TEOA was reported to be 0.87 V (vs. Ag/AgCl) [40]. Therefore, PSV˙⁺ and PSV⁰ was produced by photosensitization with ZnTPPS based on the reductive quenching process.

Fig. 9:

Difference UV-visible absorption spectrum change of the sample solution consisting of TEOA, ZnTPPS and PSV²⁺ in Bis-tris buffer (pH 7.4) with visible-light irradiation time at 30°C.

Fig. 10:

The potential diagram of ZnTPPS, TEOA and PSV²⁺.

Malate production from CO₂ and pyruvate under visible light irradiation

Figure 11 shows the time dependence of oxaloacetate (■) and malate (●) production with the system containing CO₂ and pyruvate with TEOA, H₂TPPS, PSV²⁺, Mg²⁺ and ME. When a sample solution containing TEOA, ZnTPPS, PSV²⁺, pyruvate, ME and Mg²⁺ in CO₂ saturated 10 mM bis-tris buffer (pH 7.4) was irradiated with 250 W halogen lamp, oxaloacetate and malate produced as shown in Fig. 11. In this system, the concentration of oxaloacetate increased at first 1 h irradiation, and then decreased. On the other hand, the concentration of malate increased with decreasing concentration of oxaloacetate. Oxaloacetate is formed as an intermediate, and malate is produced due to the oxaloacetate reduction. The malate concentration after 3 h irradiation was estimated to be 604 μM. The conversion yield of pyruvate to malate was 5.0%. In this system, each turnover numbers of H₂TPPS, PSV²⁺ and ME were estimated to be 5.0, 0.50 and 216 h⁻¹, respectively. Especially, the turnover number of ME was higher than those of H₂TPPS and PSV²⁺. Thus, catalytic production of malate from CO₂ and pyruvate proceeded in the system of TEOA, H₂TPPS, PSV²⁺ and ME.

Fig. 11:

Time dependence of visible light-induced oxaloacetate and malate production in the solution containing TEOA, H₂TPPS, PSV²⁺, ME, pyruvate and MgCl₂ in a CO₂ saturated bis-tris buffer (pH 7.4). (●) malate, (■) oxaloacetate.

In the absence of TEOA, H₂TPPS and PSV²⁺, oxaloacetate and malate were not observed with the system containing pyruvate, Cl⁻ and ME in CO₂ saturated bis-tris buffer (pH 7.4) under visible light irradiation. When the reaction mixture containing TEOA, H₂TPPS, PSV²⁺, pyruvate, Mg²⁺ and ME in CO₂ saturated bis-tris (pH 7.4) buffer was reacted in the dark condition, neither oxaloacetate nor malate was produced.

A proposed model for the reaction mechanism of ME is shown in Fig. 12. According to previous report [33], it is presumed that PV˙⁺ is involved in oxaloacetate reduction to malate and PV⁰ is involved in oxaloacetate production from CO₂ and pyruvate due to carbon–carbon bond formation. A couple of PSV˙⁺ causes reduction of oxaloacetate to malate with ME. The reduction potential of oxaloacetate/malate is −0.166 V (vs. NHE). As the Gibbs free energy for the oxaloacetate reduction with PSV˙⁺ oxidation was estimated to be −2.9 kJ mol⁻¹, thus this process is thermodynamically possible reaction. The photosensitization of H₂TPPS produces a PSV⁰. It is considered that a carbon–carbon bond formation from CO₂ and pyruvate was promoted by the PSV⁰. The enol type of pyruvate is required for the carbon–carbon bond formation based on the introducing CO₂ with ME in the presence of natural co-enzyme NADPH [28], [41]. It is presumed that PSV⁰ is involved in keto-enol tautomerization of pyruvate. PSV⁰ acts seemingly like a base and promotes the enolization of pyruvate with ME. Mg²⁺ stabilizes the enolate intermediate. In addition, it is assumed that PSV⁰ is acting on activation of C–H bond of pyruvate or activation of CO₂ to form oxaloacetate with ME.

Fig. 12:

Proposed mechanism for malate production from pyruvate and CO₂ via oxaloacetate with ME and multi-electron reduced PSV²⁺ (PSV˙⁺, PSV⁰).

Since reaction mechanism is a hypothesis, we will demonstrated it by using docking simulation between PSV⁰ and ME, reaction simulation of introducing CO₂ to pyruvate and crystallization of PSV⁰ and ME.

From these results, it is considered that CO₂ was introduced as a carboxy-group to pyruvate to form oxaloacetate with PSV⁰, and then reduced to malate with PSV˙⁺. By using PSV²⁺, the visible light-induced the carbon–carbon bond formation from CO₂ as a feedstock efficiency. Therefore, for the visible light-induced oxaloacetate and malate production system from CO₂ and pyruvate, the reduced form of PSV²⁺ (PSV˙⁺, PSV⁰) is essential.