Laccase [1-3] has been used as the cathode enzyme of the biofuel cell [4-7] to reduce molecular oxygen to water by four electron reaction. In order to enhance effciency of electric power generation, it is important and necessary to improve electron transfer from electrode to enzyme (in the case of laccase, T1 site  accepting electrons especially). To overcome this problem, mediators are commonly introduced between cathodeenzyme systems. Since nano-particle mediators can be involved in protein molecules, they have spatial advantage, while they also have disadvantage in view of low current density. On the other hand, metal complex mediators  have advantage in current density, though their molecular size and shape make impossible to be include into protein molecules, so long-distance electron transfer may occur generally. Therefore, metal complex mediators having spatially advantage or appropriate methods for spatial tuning have been required so far [8-10]. By the way, besides conventional application with photoisomerization , we have widely investigated organic/ inorganic hybrid materials composed of functional metal complexes and azobenzene [12, 13, 16-21, 23] in polymer films to induce optical anisotropy (namely anisotropic molecular orientation) induced by linearly polarized UV light due to Weigert effect [14, 15]. The systems have been developed to bidendate chelate chiral Schiff base complexes [12, 13], salentype chiral Schiff base complexes [16, 17], achiral complexes to induce optical anisotropy (namely anisotropic molecular orientation) induced by linearly polarized UV light due to Weigert effect [14, 15]. The systems have been developed to bidendate chelate chiral Schiff base complexes [12, 13], salentype chiral Schiff base complexes [16, 17], achiral complexes [18, 19], azo-group containing complexes [20, 21], and without azo-moiety materials (whose metal complexes have large dipole moment) [21, 22]. As a matrix, not only synthetic polymer flms but also protein flms [22, 23] were also employed and docking of metal complexes into proteins was also discussed [22, 24]. Moreover, circularly polarized UV light induced supramolecular chirality or photo-induced helical supramolecular arrangement were elucidated recently [18, 25]. Hence, polarized light induced molecular orientation may be promising method to control spatial tuning of metal complexes included in protein molecules or docking on protein surface .
Herein, we prepared organic/inorganic hybrid materials composed of chiral salen-type Schiff base Mn(II) (1), Co(II) (2), Ni(II) (3), Cu(II) (4), and Zn(II) (5) complexes (Fig. 1) and laccase. Structural characterization and docking of them will be discussed. We will elucidate to compare degree of opticalanisotropy for them caused by Weigert effect after irradiation of polarized UV light up to 10 min, which was observed by using polarized UV-vis and IR spectroscopy. Furthermore, we will attempt to observe changes of electrochemical properties of 1-5 with laccase not only potential performance as mediator but also “spatially photo-tuneable mediator” by polarized UV light irradiation to an electrode of the hybrid materials for the frst time to our knowledge.( Fig. 1)
Fig. 1. Structures of complexes 1-5 (M = Mn, Co, Ni, Cu, and Zn, respectively)
2.1. General procedures
Chemicals of the highest commercial grade available (solvents from Kanto Chemical, organic compounds from Tokyo Chemical Industry and metal sources from Wako) were used as received without further purifcation.
2.2.1. Preparations of complexes
Treatment of aniline (1.929 g 20.0 mmol) in 15 mL of 6 M HCl and NaNO 2 (1.395 g 20.0 mmol) in 30 mL of H2O for 30 min at 278 K gave rise to yellow precursor. Treatment of the precursor and o-vanillin (2.9878 g 20.0 mmol) in 30 mL of 10 % NaOH aqueous solution for 1h at 278 K, and the resulting orange precipitates of 2-hydroxy-3-methoxy- 5-phenyldiazobenzaldehyde were fltrated and washed with water and ethanol and dried for several days. To a solution of 2-hydroxy-3-methoxy-5 phenyldiazobenzaldehyde (0.5126 g, 2.00 mmol) dissolved in ethanol (60 mL), (1R,2R)-(+)-1,2- diphenylethylenediamine (0.2122 g, 1.00 mmol) was added and stirred at 323 K for 3 h to give brown solution of the ligand. Manganese(II) acetate tetrahydrate (0.2475 g, 1.00 mmol) was added to the resulting solution containing orange deposition for 3 h to give dark brown solution of the complex. This orange compound was fltered and recrystallized from chloroform/ diethylether to give single crystals for X-ray analysis (only 1, 3, and 4). Basically the other complexes were also prepared in a similar way using the corresponding metal sources.
1: Yield 0.555 g (74.47 %). Anal. Found: C, 60.73; H, 4.56; N,9.14 %. Calc. for C42H32 N6MnO4: C, 62.51; H, 4.76; N, 10.17 %. IR (KBr): 1656 cm-1 (C=N).
2: Yield 0.6686 g (88.12 %). Anal. Found: C, 62.29; H, 4.65; N,10.27 %. Calc. for C42H32 N6CoO4: C, 62.21; H, 4.74; N, 10.12 %. IR (KBr): 1658 cm-1 (C=N).
3: Yield 0.2584 g (34.66 %). Anal. Found: C, 65.60; H, 4.27; N, 10.71 %. Calc. for C42H34N6NiO4: C, 67.85; H, 4.34; N, 11.30 %. IR (KBr): 1605 cm-1 (C=N).
4: Yield 0.2785 g (33.54 %). Anal. Found: C, 63.19; H, 5.04; N, 10.41 %. Calc. for C42H36CuN6O5,C2H8O2: C, 63.49; H, 5.33; N, 10.10 %. IR (KBr) : 1621 cm-1 (C=N).
5: Yield 0.2584 g (44.68 %). Anal. Found: C, 65.33; H, 4.59; N, 10.62 %. Calc. for C42H34 N6ZnO4: C, 66.20; H, 5.05; N, 10.02 %. IR (KBr): 1658 cm-1 (C=N). 1H-NMR(CDCl3): 1.55 ppm(6H, s), 4,02 ppm(2H, s), 7.25-7.60 ppm(4H, m), 7.75 ppm(4H, m), 7.90 ppm(4H, m), 10.0 ppm(2H,s), 11.6 ppm(2H, s).
2.2.2. Preparations of hybrid materials of complex (1-5)+PMMA
Acetone solution (0.5 mL) of a complex (1-5) (0.0019 g in 5 mL acetone) and acetone solution (2 mL) of polymethylmethacrylate (PMMA) (10%) were cast onto a slide glass to give rise to a flm of hybrid materials 1+PMMA through 5+PMMA.
2.2.3. Preparations of hybrid materials of complex (1-5)+laccase
Acetone solution (0.5 mL) of a complex (1-5) (0.0019 g in 5 mL acetone) and laccase (0.0152 g in 2 mL tris buffer) were mixed and dropped onto a PMMA flm and dried for 2 days at 298 K to give rise to hybrid materials 1+laccase through 5+laccase.
2.3. Physical measurements
Elemental analyses (C, H, N) were carried out with a Perkin-Elmer 2400II CHNS/O analyzer at Tokyo University of Science. Infrared spectra were recorded as KBr pellets on a JASCO FT-IR 4200 plus spectrophotometer in the range of 4000-400 cm-1 at 298 K. Electronic spectra were measured on a JASCO V-570 UV/VIS/NIR spectrophotometer in the range of 800-200 nm at 298 K. Circular dichroism (CD) spectra were measured on a JASCO J-820 spectropolarimeter in the range of 800-200 nm at 298 K. Electrochemical (cyclic voltammetry, CV) measurements were carried out on a BAS SEC2000-UV/ VIS and ALS2323 system with Ag/AgCl electrodes range of -0.50–0.80 V vs. Ag/Ag+. 1H-NMR spectra were recorded on a JEOL JMN-300 spectrometer (300 MHz) in CDCl3. Photoillumination were carried out using a lamp (1.0 mW/cm2) with optical flters (UV λ = 200-400 nm) leading to a sample by using optical fbers and polarizer through optical flters.
2.4. X-ray crystallography
prismatic single crystals of 1 (brown), 3 (orange), and 4 (brown) were glued on top of a glass fber and coated with a thin layer of epoxy resin to measure the diffraction data. Intensity data were collected on a Bruker APEX2 CCD diffractometer with graphite monochromated Mo Kα radiation (λ = 0.71073 Å). Data analysis was carried out with a SAINT program package. The structures were solved by direct methods with a SHELXS-97  and expanded by Fourier techniques and refned by full-matrix least-squares methods based on F2 using the program SHELXL-97 . An empirical absorption correction was applied by a program SADABS . All nonhydrogen atoms were readily located and refned by anisotropic thermal parameters. All hydrogen atoms were located at geometrically calculated positions and refned using riding models. Analysis of 1 indicated partial racemization of chiral ligands during preparation. There are two crystallographically independent molecules in the asymmetric units.
Crystallographic data for 1 (CCDC 1457179). C44H39MnN6O8, crystal size 0.96 mm × 0.93 mm × 0.96 mm, Mw = 834.75, monoclinic, space group P21 (#4), a = 12.1008(15) Å, b = 16.718(2) Å, c = 23.170(3)°,β = 94.4111(16)°, V = 4673.4(10) Å3, Z = 4, Dcalc = 1.186 mg/m3, F(000) = 1540, R1 = 0.0835, wR 2 = 0.2427 (16762 reﬂections), S = 1.490, Flack parameter = 0.892(17). (where R1 =∑||Fo|-|Fc||/∑|Fo|. Rw = (∑w(|Fo|-|Fc|)2 /∑w|Fo|2)1/2, w = 1/(σ2(Fo) + (0.1P)2+0.3989P), P = (Fo + 2Fc2)/3).
Crystallographic data for 4 (CCDC 1457181). C 42H34N6NiO4, crystal size 0.07 mm × 0.06 mm × 0.05 mm, Mw= 745.46, monoclinic, space group P21 (#4), a = 12.3257(18) Å, b = 12.2272(18) Å, c = 23.418(3) Å, β = 99.206(2)°, V = 3483.9(9) Å3, Z = 4, Dcalc = 1.421 mg/m3, F(000) = 1552, R1 = 0.0384,
wR 2 = 0.1032 (10358 reﬂections), S = 0.765, Flack parameter = -0.006(13). (where R1 =∑||Fo|-|Fc||/∑|Fo|. Rw = (∑w(|Fo|-|Fc|)2 /∑w|Fo|2)1/2, w = 1/(σ2(Fo) + (0.1P)2+0.4310P), P = (Fo + 2Fc2)/3).
Crystallographic data for 5 (CCDC 1457182). C 44H35CuN6O7, crystal size 0.16 mm × 0.10 mm × 0.08 mm, Mw = 823.32, triclinic, space group P1 (#1), a = 11.4817(16) Å, b = 12.1012(17) Å, c = 15.358(2)Å, α = 93.619(2)°, β = 92.131(2)° ,γ = 107.221(2)°, V = 2030.6(5) Å3, Z = 2, Dcalc = 1.347 mg/m3, F(000) = 852, R1 = 0.0580, wR2 = 0.1568 (7457 reﬂections), S = 1.067, Flack parameter = 0.054 (17). (where R1 =∑||Fo|-|Fc||/∑|Fo|. Rw = (∑w(|Fo|-|Fc|)2 /∑w|Fo|2)1/2, w = 1/(σ2(Fo) + (0.1P)2+0.3989P), P = (Fo + 2Fc2)/3).
2.5 Computational methods
All calculations were performed using the Gaussian 09W software Revision D.01 (Gaussian, Inc.). The gas phase geometry optimizations were carried out using TD-DFT with B3LYP functional. The vertical excitation energy was calculated with the Lanl2dz for Mn, Co, Ni, Cu, Zn and with the 6-31+G(d) basis set for H, C, N and O method based on the singlet ground state geometry .
3. Results and discussion
3.1. Crystal structures of 1, 4, and 5
The molecular structures of 1, 4, and 5 are depicted in Figs. 2-4, respectively. Complexs 1, 4, and 5 afford a sixcoordinated octahedral [MnN2O2], four-coordinated squareplanar cis-[NiN2O2], and fve-coordinated square pyramidal [CuN2O3] coordination geometries having 2, 0, and 1 water molecules as axial ligands. Selected bonds are written in each figure caption. In all crystals, azo-groups afford a trans confguration, and λ-confguration in diamine ligands. Most of them are within normal values for the analogous Schiff base metal complexes [30-32]. The molecular packing in solid state is essentially formed by weak van der Waals forces predominantly.
Fig. 2. Crystal structures of 1 showing selected atom labeling scheme. Hydrogen atoms are omitted for clarity. Selected bond distances (Å) are Mn1-O15=1.884(6), Mn1-O16=1.916(7), Mn1-N13=1.964(8), Mn1-N7=1.987(7), Mn1-O21=2.116(9), Mn1-O13=2.375(7), Mn2-O6=1.885(6), Mn2-O5=1.867(8), Mn2-N1=1.987(7), Mn2-N2=2.024(9), Mn2-O7=2.117(8), Mn2-O8=2.353(7).
3.2. Docking of 1-5 into laccase
Firstly, in order to estimate solution structures of complexes, experimental (acetone solutions) and simulated (based on TD-DFT optimized structures) UV-vis and CD spectra of 1-5 were compared as shown in Fig. 5. Qualitatively, experimental spectra were reasonably reproduced and assigned by simulated ones generted according to the literature procedures . For example, intense π−π* peaks of 1-5 appeared at 430, 431, 413, 434, and 407 nm, respectively, in actual solutions, while the corresponding simulated peaks of 1-5 appeared at 430, 431, 413, 434, and 407 nm, respectively. The simulated CD spectra of the complexes by TD-DFT support that a certain supramolecular arrangement was induced in chiral laccase matrix by irradiation of linearly polarized UV light. Predominant absorption band of electronic transition (419 nm, HOMO-1(189) to LUMO+3(191)) will be also associated increase in current density of 1observed experimentally (see later section).
Secondly, difference of CD spectra of solutions indicated keeping secondary structure (exhibiting random coil and α-helix band at 195-220 nm generally ) of laccase after docking a complex 5 (without α-helix band) as to be 5+laccase regardless of CD intensity emerged by chiral 5. Moreover, gradual change of UV-vis spectra with increasing amount of 5 added was also exhibited characteristic band shift around 400 nm (Fig. 6). Both spectral features suggested that a hybrid material (5+laccase) is quantatively formed by intermolecular interaction between laccase and 5 at a certain surface region of laccase molecules without structural changes of peptide chains.
Thirdly, computational docking simulation using GOLD suite  with PDB 1GYC structure of this laccase  was performed (Fig. 7). Judging from recent standard , the best ftting score was obtained by assuming including a complex (5) in a pocket region around T1 site binding by hydrogen bonds or aromatic π−π interaction. Some nearest intermolecular distances are C1(Phe285 of laccase) – O1(5) = 0.2818 nm, C2(Phe285) – H1(5) = 0.2381 nm, C3(Phe285) – C4(5) = 0.2796 nm, H2(Phe285) – C5(5) = 0.2286 nm, H3(Gly392) – H4(5) = 0.1948 nm, H3(Gly382) – C8(5) = 0.2932 nm, H5(Gly392) – H6(5) = 0.1718 nm.
Fig. 3. Crystal structures of 4 showing selected atom labeling scheme. Hydrogen atoms are omitted for clarity. Selected bond distances (Å) are Ni1-N3=1.837(4), Ni1-O4=1.837(3), Ni1-N6=1.857(3), Ni1-O2=1.860(3), Ni2-O6=1.842(3), Ni2-O8=1.843(3), Ni2-N9=1.850(3), Ni2-N12=1.862(4)
Fig. 4. Crystal structures of 5 showing selected atom labeling scheme. Hydrogen atoms are omitted for clarity. Selected bond distances (Å) are Cu1-O2=1.909(6), Cu1-O4=1.920(6), Cu1-N3= 1.923(7), Cu1-N6=1.939(7), Cu1-O9=2.387(6), Cu2-N9=1.931(7), Cu2-O8 1.934(6), Cu2-O6=1.940(6), Cu2-N12=1.985(7), Cu2-O10=2.443(6).
Fig. 5. Experimental (acetone solutions; black) and simulated (TD-DFT; red) UV-vis and CD spectra based on TD-DFT optimized structures of 1-5 with electronic distribution
Fig. 6. (a) CD spectra of 5 (red), laccase (green), and 5+laccase (blue) in acetone solutions. (b) UV-vis spectra of 5 (red), laccase and one equivalent of 5 (green), laccase and two equivalent of 5 (purple), and laccase and three equivalent of 5 (blue)
Fig. 7. (left) Intermolecular interaction around docking residues (right) overall location of docking simulation by GOLD of laccase and a complex.
3.3. Polarized UV-vis and IR spectra after linearly polarized UV light irradiation
Fig. 8 depicts polarized UV-vis absorption spectra and the corresponding circular diagrams of angular dependence of absorbance of π−π*, n−π*, and d-d bands at 270, 335, and 685 nm, respectively, for 1+laccase (as a cast flm) after linearly polarized UV light (< 350 nm) irradiation up to 10min. Besides drastic change of π−π* and n−π*, bands ue to photoisomerization of azobenzene moiety from trans to cis , the optical anisotropy of (cis-)azobenzene compound increased because of Weigert effect (molecular reorientation with dipole moment of (cis-)azobenzene to be perpendicular to the electric vector of linearly polarized UV light) , which was also observed in other bis-bidentate Schiff base metal complexes having azobenzene moiety even in laccase matrix (forming hybrid material by regularly docking) . Although UV-vis spectra indicated overlapped information of a complex and laccase especially in UV region, polarized IR spectra (reading C=N and N=N bands) can observe separated information. For all systems, parameters derived from UV-vis and IR spectra reasonably indicated increasing such anisotropy (Tables S1-S4).
Table S1. The R values from polarized UV-vis spectra of for 1 through 5+laccase after polarized UV light irradiation.
Table S2. The S values from polarized UV-vis spectra of for 1 through 5+laccase after polarized UV light irradiation.
Table S3. The R values from polarized IR spectra of for 1 through 5+laccase after polarized UV light irradiation.
Table S4. The S values from polarized IR spectra of for 1 through 5+laccase after polarized UV light irradiation
3.4. Electrochemical behavior and photo-induced molecular orientation of a complex
In order to investigate novel performance of 1-5 as mediator to laccase on RRDE  as the fnal goal (Fig. 9), frstly redox behavior of 1-5 before and after UV light irradiation was confrmed (Fig. 10). As known well, cis-trans photoisomerization of azobenzene moiety of ligand results in slight changes of both redox potentials and current densities for all 1-5. Therefore, the cis form and its redox potential should be assumed for species after UV light irradiation. Secondly, comparison between laccase and hybrid materials of a complex and laccase was investigated (Fig. 11). Even keeping trans form without UV light condition, current density predominantly increased for 1-4, while complicated behavior could be observed due to redox reaction by ligand for 5. Therefore, complexes (1-4, especially 1) could act as a good mediator for laccase by formation of hybrid materials. Thirdly, differences of CV before and after polarized UV light irradiation, which was expected to lead to not only cis-trans photoisomerization but also anisotropic molecular orientation, were compared for hybrid materials 1 through 5 + laccase (Fig. 9). Based on examination above, shift of redox potential mainly results from cis-trans photoisomerization , while increasing current density mainly can be regarded as improving performance as a mediator. Although 2+laccase, 3+laccase, 5+laccase indicated only slight difference, 1+laccase and 4+laccase indicated drastic changes of CV features. As for 4+laccase, a new peak emerged at about -0.5 V, which can be attributed to additional redox by ligand moiety except for original metal ion moiety. On the other hand, as for 1+laccase, increasing current density without shift of redox potentials could be observed, which can be ascribed to improve performance as a mediator to laccase. Its reason is molecular orientation of cis form 1 in the pocket of laccase molecular surface. As elucidated by docking simulation, it is fne tuning of molecular orientation of 1 to form suitable hydrogen bonding pathway between 1 and laccase to conduct electron [37, 38].
Fig. 9. CV (scan rate 0.05 Vs-1) measured using RRDE electrodes for Nafon coated hybrid materials 1 through 5+laccase after polarized UV light for 0 (black) and 10 (red) min.
Fig. 10. CV (scan rate 0.05 Vs-1) measured using RRDE electrodes for Nafon coated a complex 1-5 after UV light irradiation for 0 (black) and 10 (red) min to confrm effects from trans to cis photoisomerization of a complex.
Fig. 11. CV (scan rate 0.05 Vs-1) measured using RRDE electrodes for Nafon coated laccase (green), a complex 1-5 (blue), and hybrid materials 1 through 5+laccase (red) to confrm effects by formation of hybrid materials.
In summary, we prepared fve chiral Schiff base metal complexes whose ligand incorporate azobenzene moiety, which can exhibit Weigert effect (anisotropic molecular orientations) by polarized UV light irradiation. These complexes can bind to laccase in the pocket on the surface near T1 site. By polarized UV light irradiation to 1+laccase, it indicated novel increasing current density, which is attributed to molecular orientation tuning in laccase. In order for application cathode material for biofuel cell, this photo-tuneable mediator will be a new promising molecular design strategy. Further study on this method and the analogous materials are in progress now.
The computations were performed using Research Centre for Computational Science, Okazaki, Japan