A Novel Photo-Driven Hydrogenation Reaction of an NAD+-Type Complex Toward Artificial Photosynthesis

The photocatalytic reduction of carbon dioxide (CO2) to value-added chemicals is an attractive strategy to utilize CO2 as a feedstock for storing renewable energy, such as solar energy, in chemical bonds. Inspired by the biological function of the nicotinamide adenine dinucleotide redox couple (NAD+/NADH), we have been developing transition-metal complexes containing NAD+/NADH-functionalized ligands to create electro- and/or photochemically renewable hydride donors for the conversion of CO2 into value-added chemicals. Our previous findings have provided insights for the development of photocatalytic organic hydride reduction reactions for CO2, however, further examples, as well as investigation, of these photo-driven NAD+/NADH-type hydrogenation and organic hydride transfer reactions are required not only to explore the mechanism in detail but also to develop a highly efficient catalyst for artificial photosynthesis. In this paper, we report the synthesis, characterization, and photo-induced NAD+/NADH conversion properties of a new ruthenium(II) complex, [Ru(bpy)2(Me-pn)](PF6)2 (1), which contains a new NAD+-type ligand, Me-pn (2-methyl-6-(pyridin-2-yl)-1,5-naphthyridine). In addition, we have succeeded in the isolation of the corresponding two-electron reduced ruthenium(II) complex containing the NADH-type ligand Me-pnHH (2-methyl-6-(pyridin-2-yl)-1,4-dihydro-1,5-naphthyridine), i.e., [Ru(bpy)2(Me-pnHH)](PF6)2 (1HH), by the photo-induced hydrogenation reaction of 1. Thus, in this study, a new photo-driven NAD+/NADH-type hydrogenation reaction for possible CO2 reduction using the NAD+/NADH redox function has been constructed.


INTRODUCTION
Currently, the design and development of novel visible-light photoredox catalysts for carbon dioxide (CO 2 ) reduction are considered to be crucial challenges. In particular, it is vital not only to clarify how to employ solar energy, which is a sustainable clean energy source, but also to develop methods to convert CO 2 , which is a major greenhouse gas and is harmful for the environment, into chemical fuels and feedstocks in the viewpoint of the current global energy and environmental crisis (Fox and Dulay, 1993;Kisch, 2013). Many research efforts have so far been devoted to the search for new and efficient catalysts for CO 2 conversion into value-added chemicals such as carbon monoxide (CO), formic acid (HCO 2 H), and methanol (CH 3 OH) (Meyer, 1989;Alstrum-Acevedo et al., 2005;Wang et al., 2015).
However, there are several problems facing photochemical CO 2 conversion, particularly related to the side reactions and low selectivity toward specific reduction products, as well as poor energy efficiency (Leitner, 1995;Jones et al., 2014). To solve these problems, the use of transition-metal coordination compounds as photocatalysts for CO 2 reduction has drawn significant attention because some of these compounds exhibit significant photocatalytic activity for CO 2 reduction (Morris et al., 2009;Berardi et al., 2014), and they play an essential role in artificial photosynthesis (Fukuzumi et al., 2018), as well as natural photosynthesis (Silva et al., 2015).
In particular, ruthenium complexes are promising candidates for photocatalysts because most Ru complexes show excellent photophysical properties with relatively long excited-state lifetimes and visible light absorption bands originating from metal-to-ligand charge transfer (MLCT) (Medlycott and Hanan, 2005;Sun et al., 2015;Dongare et al., 2017). In the past several decades, a number of Ru complexes having photocatalytic ability toward CO 2 reduction have been reported (Jessop et al., 1995;Tanaka and Ooyama, 2002;Kuramochi et al., 2018). In this context, we have focused on Ru complexes having NAD + /NADH-functionalized ligands because the biological function of the NAD + /NADH redox couple is as a generator and reservoir of hydride ions (H − ), which are equivalent to two electrons and one proton (Eisner and Kuthan, 1972;Walsh, 1980;Stout and Meyers, 1982;Gebicki et al., 2004;Bilan et al., 2015), and is of great interest for the development of photorenewable hydride reagents.

Materials
All chemicals used for the synthesis of the ligands and complexes were commercial products of the highest available purity and were used without further purification unless otherwise indicated. Solvents were purified by standard methods before use (Armarego and Chai, 2009).

Synthesis
All ligands and complexes used in this study were prepared according to the following procedures and the structures of the products were confirmed from analytical data (vide infra).

Physical Measurements
All manipulations were carried out under an argon atmosphere using standard Schlenk techniques or in a glovebox. The absorption spectra were obtained using a Hewlett-Packard 8453 diode array spectrophotometer or a Shimadzu UV-3100PC scanning spectrophotometer. The emission spectra were recorded on a JASCO FP-8500 spectrofluorometer. The emission lifetimes were performed with a UNISOKU LSP-1000N-PX spectrometer. The electrospray (ESI)-mass spectrometry (MS) data were obtained with a Shimadzu LCMS-2020. Elemental analyses were performed using a Yanaco CHN Corder MT-5 (A Rabbit Science Japan Co., Ltd.). NMR measurements were measured with a JEOL JMN-ECX 300 (300 MHz) NMR spectrometer. Electron spin resonance (ESR) spectra were taken with a JEOL JES-X310 equipped with a variable temperature (VT) apparatus and were recorded under non-saturating microwave power conditions. The magnitude of the modulation was chosen to optimize the resolution and the signal-to-noise ratio of the observed spectra. The g values were calibrated with a Mn(II) marker used as a reference. Cyclic voltammetry measurements were performed on an ALS/Chi model 733D electrochemical analyzer in a deaerated solvent containing 0.1 M tetra-nbutylammonium hexafluorophosphate (TBAPF 6 ) as a supporting electrolyte. A conventional three-electrode cell was used with a glassy-carbon working electrode and a platinum wire as the counter electrode. The glassy-carbon working electrode was routinely polished with a BAS polishing alumina suspension and rinsed with acetone before use. The reversibility of the electrochemical processes was evaluated by standard procedures, and all potentials were recorded against an Ag/AgCl reference electrode. All electrochemical measurements were carried out under an argon atmosphere.

X-ray Crystal Structure Determination
The single crystal X-ray diffraction data of 1•CH 3 CN•Et 2 O were collected on a Rigaku VariMax RAPID-DW/NAT with Mo-Kα radiation (λ = 0.71075 Å) at 173 K and processed using RapidAuto program (Rigaku). An empirical absorption correction resulted in acceptable transmission factors. The data were corrected for Lorentz and polarization factors. The structure was solved by direct methods using SHELXT-2018/2 (Sheldrick, 2015) and refined by full-matrix least-squares techniques on F 2 using SHELXL-2018/3 (Sheldrick, 2015). All non-hydrogen atoms were refined anisotropically and all hydrogen atoms were included in the refinement process as per the riding model. CCDC-1894384 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Center, 12, Union Road, Cambridge CB21EZ, UK; fax: (+44)1223-336-033; or deposit@ccdc.cam.ac.uk).
Well-shaped single crystals of 1 suitable for X-ray structure determination were obtained by recrystallization from an acetonitrile (CH 3 CN) solution using the slow vapor diffusion of diethylether (Et 2 O). This compound crystallizes in the monoclinic P2 1 /n space group with four molecules in the unit cell. The structure also contains solvated CH 3 CN and Et 2 O for each complex. The molecular structure of 1 is shown in Figure 1. The ruthenium(II) ion in 1 has a hexacoordinate octahedral structure formed of four N atoms of the two bpy ligands and two N atoms of the Me-pn ligand. The coordination environment of 1 is almost the same as that of a previously reported ruthenium(II) NAD + -type complex [Ru(bpy) 2 (pbn)](PF 6 ) 2 (Koizumi and Tanaka, 2005), and the bond lengths between the ruthenium(II) ion and the six N atoms of the ligands for 1 (2.046(4) to 2.119(4) Å) are not only the same as those of [Ru(bpy) 2 (pbn)](PF 6 ) 2 (2.038(5) to 2.116(5) Å) (Koizumi and Tanaka, 2005) but also within the typical range of those of reported ruthenium(II) complexes having a similar coordination environment (Fukushima et al., 2009(Fukushima et al., , 2010Ohtsu and Tanaka, 2012a;Kobayashi et al., 2016). The ESI-MS results for 1 in CH 3 CN show a dominant signal at m/z = 780, as shown in Figure S1, and the observed mass value, as well as the isotopic pattern, agree well with those of the simulated pattern of [Ru(bpy) 2 (pbn)](PF 6 )] + . In addition, the 1 H-NMR spectrum of 1 in CD 3 CN ( Figure S2) displays a spectral pattern reflecting the Cs symmetry of 1, including 24 aromatic proton signals between 7.11 and 8.73 ppm and three proton singlet signal assigned to the methyl group at 2.62 ppm. These results are in complete agreement with the single-crystal X-ray diffraction analysis described above.
The absorption and emission spectra of 1 in CH 3 CN at 298 K are shown in Figure 2. Characteristic absorption bands centered around 440 and 480 nm can be assigned to MLCT transitions from the ruthenium(II) centers to the bpy ligand (Caspar and Meyer, 1983) and the Me-pn ligand, respectively. Complex 1 also exhibits a 3 MLCT emission band around 685 nm upon excitation at 480 nm, as shown in Figure 2. Additionally, the emission lifetime has been evaluated from the resulting single exponential emission decay (368 ns, see Figure S3), and this emission lifetime is larger than that of a previously reported ruthenium(II) NAD +type complex [Ru(bpy) 2 (pbn)](PF 6 ) 2 (140 ns) (Polyansky et al., 2007). Furthermore, the emission quantum yield (λ ex = 480 nm) was estimated at ø = 0.37% using a relative method (Eaton, 1988) with [Ru(bpy) 3 ](PF 6 ) 2 as a reference (0.095) (Suzuki et al., 2009;Ishida et al., 2010). This value is more than 500 times larger than that of [Ru(bpy) 2 (pbn)](PF 6 ) 2 (0.00071%) (Ohtsu and Tanaka, 2012a) reported previously. Figure 3A shows the cyclic voltammogram (CV) of 1 (1 mM) in dimethylformamide (DMF) at 298 K. The CV shows one reversible redox wave at E 1/2(a) = 1.42 V (vs. Ag/AgCl) in the range above the rest potential (E rest = 0.00 V) and three reversible redox waves at E 1/2(b) = −0.88 V, E 1/2(c) = −1.31 V, and E 1/2(d) = −1.55 V in the range below E rest = 0.00 V. The E 1/2(a) redox wave can be assigned to the Ru(II)/Ru(III) couple on the basis of the rest potential. To clarify the assignment of the other three redox waves, the CV of 1 upon the addition of an excess amount of a proton source such as acetic acid (AcOH) was measured in accordance with the previously reported procedure (Kobayashi et al., 2016). The CV changes observed upon the addition of 10 equivalents of AcOH into a solution of 1 in DMF are shown in Figure 3B. As shown, the reversibility of the E 1/2(b) redox wave changes drastically to an irreversible cathodic peak at E ′ pc(b) = −0.92 V, and a new anodic peak at E ′ pa(b) = 0.49 V coupled with an irreversible E ′ pc(b) peak appears, whereas the other two E 1/2(c) and E 1/2(d) redox waves are barely changed from the redox waves at E ′ 1/2(c) = −1.19 V and E ′ 1/2(d) = −1.41 V, respectively, maintaining redox reversibility. The lack of reversibility of the E 1/2(b) redox wave in the presence of the proton source is most likely caused by the protonation of the non-bonded N atom of the reduced Me-pn ligand; thus, the redox wave at E 1/2(b) = −0.88 V can be assigned to the Mepn/Me-pn •− couple. The large separation between the cathodic and anodic peak potentials of the Me-pn localized redox reaction under protic conditions may result from significant structural changes, such as the hydrogenation of the NAD + -type Me-pn ligand to form the NADH-type Me-pnHH ligand through a two electron and two proton process. In the case of the other two redox waves (E 1/2(c) = −1.31 V and E 1/2(d) = −1.55 V), it is safe to conclude that the E 1/2(c) and E 1/2(d) redox waves correspond to the redox couples of the two bpy ligands since almost no changes in the reversibility and the position of these redox waves are observed upon the addition of 10 equivalents of AcOH on the basis of CV measurements described above. In addition, the pK a of the Me-pn ligand in 1 has been determined by spectrophotometric acid-base titration from pH 0.29 to pH 4.65, as shown in Figure S4. The estimated pK a value of the protonated species [Ru(bpy) 2 (Me-pnH)] 3+ is 1.6, the value of which is almost the same as previously reported in the case of [Ru(bpy) 2 (pbn)](PF 6 ) 2 (pK a = 1.7) (Polyansky et al., 2007).
In the absence of TEOA, complex 1 in CH 3 CN was quite stable even when exposed to visible light (150-W Xe lamp, λ > 420 nm) (see Figure S5). However, absorption spectral changes were observed upon the irradiation with visible light to a solution of 1 in the presence of TEOA (CH 3 CN:TEOA = 4:1 v/v) at 293 K as shown in Figure 4. This reaction consists of two distinct steps. First, the characteristic absorption band located at 480 nm arising from 1 (red line) rapidly changes, and a spectrum (purple line) exhibiting a new broad band around 1,000 nm with an accompanying isosbestic point at 448 nm from 0 to 20 s is obtained; in addition, the intensity of the spectrum  Frontiers in Chemistry | www.frontiersin.org around 1,000 nm subsequently decreased. This is accompanied by an increase in the absorption band located at 420 nm (orange line) with an isosbestic point at 477 nm from 20 to 900 s. When triethylamine (TEA, E ox = 0.66 V vs. SCE) (Yanagida et al., 1997), which is also a sacrificial reagent, was used instead of TEOA, almost the same absorption spectral changes indicating a twostep process have been observed (Figure S6), and a dominant signal (m/z = 782), which agrees well with the simulated pattern corresponding to the NADH-type complex, [Ru(bpy) 2 (Me-pnHH)](PF 6 )] + , has been detected by ESI-MS measurements of the final reaction mixture, as shown in Figure S7. Furthermore, attempts to isolate the NADH-type complex [Ru(bpy) 2 (Me-pnHH)](PF 6 ) 2 (1HH) by photochemical reduction using TEOA were successful based on the ESI-MS at m/z = 782 and the observed mass value, as well as the isotopic pattern, which agrees well with the simulated pattern of [Ru(bpy) 2 (Me-pnHH)](PF 6 )] + . In addition, 22 characteristic proton signals in the aromatic region, a proton N-H signal at 6.68 ppm, and two proton signals at 3.36 and 2.35 ppm, which can be assigned to the methylene protons of the 1,4-dihydropyridine frameworks in the Me-pnHH ligand (see Material and Methods and Figure S8), were observed. As reported for the previous ruthenium(II) NAD + -type complex [Ru(bpy) 2 (pbn)] 2+ , [Ru(bpy) 2 (pbn)] 2+ is efficiently reduced to the corresponding NADH-type complex ([Ru(bpy) 2 (pbnHH)] 2+ ) by photochemical reduction in CH 3 CN in the presence of a sacrificial electron donor such as TEOA or TEA. The absorption spectral changes for the photo-driven NAD + /NADH-type hydrogenation reaction in [Ru(bpy) 2 (pbn)] 2+ system were observed in a one-step process (Ohtsu and Tanaka, 2012a) despite the use of the same experimental conditions as those of 1 (150-W Xe lamp(λ > 420 nm), CH 3 CN:TEOA = 4:1 v/v, 293 K). Such a difference in the reactivity may be ascribed to the steric effects of the methyl group of the Me-pn ligand in 1, which result in weaker π-π interactions between two [Ru(bpy) 2 (Me-pnH • )] 2+ (1 •− H) radical intermediate species compared to the case of the nonsubstituted pbn ligand in [Ru(bpy) 2 (pbn)] 2+ ; this is based on a possible mechanism for the photo-driven NAD + /NADH-type hydrogenation reaction discussed later.
To gain further mechanistic insight into the photoinduced NAD + /NADH-type hydrogenation reaction of 1, ESR measurements after photoirradiation for 20 and 900 s under the same conditions as those used to observe the absorption spectral changes shown in Figure 4 were carried out (Figure 5). The ESR spectrum of 1 in CH 3 CN:TEOA = 4:1 v/v solution after photoirradiation for 20 s at 173 K displays a pseudo-isotropic signal at g = 2.000 with a line width of H = 19 G, as shown in Figure 5A. In contrast to the case of photoirradiation for 900 s, no ESR signal is observed (Figure 5B). The observed ESR signal in the case of photoirradiation for 20 s (Figure 5A) is similar to that of a one electron reduced [Ru(bpy) 3 ] 2+ complex, namely [Ru(bpy) 2 (bpy •− )] + (g = 1.996 and H = 26 G at 173 K) (Motten et al., 1981), which can be assigned to a ligand-centered radical species [Ru(bpy) 2 (Me-pnH • )] 2+ (1 •− H) based on a previous mechanistic study of the conversion of [Ru(bpy) 2 (pbn)] 2+ into [Ru(bpy) 2 (pbnHH)] 2+ (Polyansky et al., 2008). Complex 1HH, which is the photoreduction product of 1 in the presence of a sacrificial reagent, is diamagnetic, which is consistent with the results of the ESR measurements in the case of photoirradiation for 900 s.
Based on the various experimental results described above and the previously reported mechanism for the photodriven NAD + /NADH-type hydrogenation reaction using [Ru(bpy) 2 (pbn)] 2+ (Polyansky et al., 2008), a photo-driven hydrogenation reaction pathway of 1 into 1HH in the presence of a sacrificial reagent under photoirradiation can be proposed, SCHEME 1 | A possible reaction mechanism for the photo-driven NAD + /NADH-type hydrogenation reaction from 1 to 1HH.
Frontiers in Chemistry | www.frontiersin.org as shown in Scheme 1. First, the NAD + -type complex 1 is photochemically reduced to the Me-pn radical anion species [Ru(bpy) 2 (Me-pn •− )] + (1 •− ) by the reductive quenching of the photoexcited 1 * by a sacrificial donor such as TEOA or TEA (step I). Secondly, the protonation of the non-coordinating N atom of the Me-pn •− in 1 •− produces [Ru(bpy) 2 (Me-pnH • )] 2+ (1 •− H) radical species (step II). Then, each 1 •− H dimerizes via the π-π stacking of the neutral Me-pnH • moieties to afford {[Ru(bpy) 2 (Me-pnH • )] 2+ } 2 ((1 •− H) 2 ) (step III). Finally, intramolecular proton-coupled electron transfer from one Me-pnH • in (1 •− H) 2 to another leads to disproportionation, thus generating the 1HH NADH-type complex and regenerating the 1 NAD + -type complex. At present, we have yet to clarify the reason why there is a significant difference in the photo-driven NAD + /NADH-type hydrogenation reaction between 1 and the previously reported [Ru(bpy) 2 (pbn)] 2+ system. However, the steric hindrance of the methyl groups in Me-pn complex may be the reason why we can see the radical intermediate species such as 1 •− H in the ESR measurements more easily than for the previously reported [Ru(bpy) 2 (pbn)] 2+ .

CONCLUSIONS
In conclusion, the present study has demonstrated the synthesis, characterization, and photoinduced NAD + /NADH conversion properties of a new ruthenium(II) complex, [Ru(bpy) 2 (Mepn)](PF 6 ) 2 (1), as well as the isolated NADH-type complex [Ru(bpy) 2 (Me-pnHH)](PF 6 ) 2 (1HH) under photochemical reduction conditions. The findings of the present study suggest a new type of photo-driven NAD + /NADH-type hydrogenation reaction that makes possible the development of photoinduced CO 2 reduction reactions utilizing the NAD + /NADH redox function. These results provide valuable information for the further development of functional transition-metal NAD + /NADH-type complexes and shed new light on the applications of the NADH-type complexes for future catalysts and energy storage materials in the area of artificial photosynthesis. Further investigations concerning photochemical reduction of CO 2 and other substrates by using the NAD + /NADH-functionalized complexes are now in progress.

DATA AVAILABILITY
The datasets generated for this study can be found in Cambridge Crystallographic Data Center, CCDC 1894384.

AUTHOR CONTRIBUTIONS
HO directed the project, conceived and designed the experiments, supervised the progress of this work, and wrote the manuscript. TS carried out the syntheses of ligands and complexes and performed most of the experiments. KT contributed X-ray structural analyses. All authors listed have discussed the results, drawn conclusions, and given the approval to the final version of the manuscript.